Oligonucleotides having A-DNA form and B-DNA form conformational geometry

ABSTRACT

Modified oligonucleotides containing both A-form conformation geometry and B-from conformation geometry nucleotides are disclosed. The B-form geometry allows the oligonucleotide to serve as substrates for RNase H when bound to a target nucleic acid strand. The A-form geometry imparts properties to the oligonucleotide that modulate binding affinity and nuclease resistance. By utilizing C2′ endo sugars or O4′ endo sugars, the B-form characteristics are imparted to a portion of the oligonucleotide. The A-form characteristics are imparted via use of either 2′-O-modified nucleotides that have 3′ endo geometries or use of end caps having particular nuclease stability or by use of both of these in conjunction with each other.

CROSS-REFERENCE OF RELATED APPLICATIONS

This Application is a continuation-in-part of Ser. No. 09/303,586, filedMay 3, 1999 now U.S. Pat. No. 6,369,209 and of Ser. No. 08/936,166,filed Sep. 23, 1997 now U.S. Pat. No. 6,307,040. application Ser. No.08/936,166 is a divisional of Ser. No. 07/835,932, filed Mar. 5, 1992,now U.S. Pat. No. 5,670,633, which derives from International PatentApplication Serial No. PCT/US91/05720, filed Aug. 12, 1991 and publishedas WO 92/03568 on Mar. 5, 1992. Each of the foregoing applications isincorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present invention relates to oligonucleotides that have both A-formand B-form conformational geometry and methods of using sucholigonucleotides. The oligonucleotides of the invention are useful intherapeutic and investigative purposes. More specifically, the presentinvention is directed to oligonucleotides having particularmodifications that will increase affinity and nuclease resistance whileconcurrently serving as substrates for RNase H when bound to a targetRNA strand.

BACKGROUND OF THE INVENTION

It is well known that most of the bodily states in mammals, includingmost disease states, are affected by proteins. Classical therapeuticmodes have generally focused on interactions with such proteins in aneffort to moderate their disease-causing or disease-p otentiatingfunctions. However, recently, attempts have been made to moderate theactual production of such proteins by interactions with molecules thatdirect their synthesis, such as intracellular RNA. By interfering withthe production of proteins, maximum therapeutic effect and minimal sideeffects may be realized. It is the general object of such therapeuticapproaches to interfere with or otherwise modulate gene expressionleading to undesired protein formation.

One method for inhibiting specific gene expression is the use ofoligonucleotides. Oligonucleotides are now accepted as therapeuticagents. A first such oligonucleotide has been approved for humantherapeutic use by the FDA and is available in commercial marketplace.

Oligonucleotides are known to hybridize to single-stranded DNA or RNAmolecules. Hybridization is the sequence-specific base pair hydrogenbonding of nucleobases of the oligonucleotide to the nucleobases of thetarget DNA or RNA molecule. Such nucleobase pairs are said to becomplementary to one another. The concept of inhibiting gene expressionthrough the use of sequence-specific binding of oligonucleotides totarget RNA sequences, also known as antisense inhibition, has beendemonstrated in a variety of systems, including living cells (forexample see: Wagner et al., Science (1993) 260: 1510-1513; Milligan etal., J. Med. Chem., (1993) 36:1923-37; Uhlmann et al., Chem. Reviews,(1990) 90:543-584; Stein et al., Cancer Res., (1988) 48:2659-2668).

The events that provide the disruption of the nucleic acid function byantisense oligonucleotides (Cohen in Oligonucleotides: AntisenseInhibitors of Gene Expression, (1989) CRC Press, Inc., Boca Raton, Fla.)are thought to be of two types. The first, hybridization arrest, denotesthe terminating event in which the oligonucleotide inhibitor binds tothe target nucleic acid and thus prevents, by simple steric hindrance,the binding of essential proteins, most often ribosomes, to the nucleicacid. Methyl phosphonate oligonucleotides: Miller, P. S. and Ts'O, P. O.P. (1987) Anti-Cancer Drug Design, 2:117-128, and α-anomeroligonucleotides are the two most extensively studied antisense agentswhich are thought to disrupt nucleic acid function by hybridizationarrest.

The second type of terminating event for antisense oligonucleotidesinvolves the enzymatic cleavage of the targeted RNA by intracellularRNase H. A 2′-deoxyribofuranosyl oligonucleotide or oligonucleotideanalog hybridizes with the targeted RNA and this duplex activates theRNase H enzyme to cleave the RNA strand, thus destroying the normalfunction of the RNA. Phosphorothioate oligonucleotides are probably themost prominent example of an antisense agent that operates by this typeof antisense terminating event.

Oligonucleotides may also bind to duplex nucleic acids to form triplexcomplexes in a sequence specific manner via Hoogsteen base pairing (Bealet al., Science, (1991)251:1360-1363; Young et al., Proc. Natl. Acad.Sci. (1991) 88:10023-10026). Both antisense and triple helix therapeuticstrategies are directed towards nucleic acid sequences that are involvedin or responsible for establishing or maintaining disease conditions.Such target nucleic acid sequences may be found in the genomes ofpathogenic organisms including bacteria, yeasts, fungi, protozoa,parasites, viruses, or may be endogenous in nature. By hybridizing toand modifying the expression of a gene important for the establishment,maintenance or elimination of a disease condition, the correspondingcondition may be cured, prevented or ameliorated.

In determining the extent of hybridization of an oligonucleotide to acomplementary nucleic acid, the relative ability of an oligonucleotideto bind to the complementary nucleic acid may be compared by determiningthe melting temperature of a particular hybridization complex. Themelting temperature (T_(m)), a characteristic physical property ofdouble helices, denotes the temperature (in degrees centigrade) at which50% helical (hybridized) versus coil (unhybridized) forms are present.T_(m) is measured by using the UV spectrum to determine the formationand breakdown (melting) of the hybridization complex. Base stacking,which occurs during hybridization, is accompanied by a reduction in UVabsorption (hypochromicity). Consequently, a reduction in UV absorptionindicates a higher T_(m). The higher the T_(m), the greater the strengthof the bonds between the strands.

Oligonucleotides may also be of therapeutic value when they bind tonon-nucleic acid biomolecules such as intracellular or extracellularpolypeptides, proteins, or enzymes. Such oligonucleotides are oftenreferred to as ‘aptamers’ and they typically bind to and interfere withthe function of protein targets (Griffin, et al., Blood, (1993),81:3271-3276; Bock, et al., Nature, (1992) 355: 564-566).

Oligonucleotides and their analogs have been developed and used fordiagnostic purposes, therapeutic applications and as research reagents.For use as therapeutics, oligonucleotides must be transported acrosscell membranes or be taken up by cells, and appropriately hybridize totarget DNA or RNA. These critical functions depend on the initialstability of the oligonucleotides toward nuclease degradation. A seriousdeficiency of unmodified oligonucleotides which affects theirhybridization potential with target DNA or RNA for therapeutic purposesis the enzymatic degradation of administered oligonucleotides by avariety of intracellular and extracellular ubiquitous nucleolyticenzymes referred to as nucleases. For oligonucleotides to be useful astherapeutics or diagnostics, the oligonucleotides should demonstrateenhanced binding affinity to complementary target nucleic acids, andpreferably be reasonably stable to nucleases and resist degradation. Fora non-cellular use such as a research reagent, oligonucleotides need notnecessarily possess nuclease stability.

A number of chemical modifications have been introduced intooligonucleotides to increase their binding affinity to target DNA or RNAand resist nuclease degradation.

Modifications have been made to the ribose phosphate backbone toincrease the resistance to nucleases. These modifications include use oflinkages such as methyl phosphonates, phosphorothioates andphosphorodithioates, and the use of modified sugar moieties such as2′-O-alkyl ribose. Other oligonucleotide modifications include thosemade to modulate uptake and cellular distribution. A number ofmodifications that dramatically alter the nature of the internucleotidelinkage have also been reported in the literature. These includenon-phosphorus linkages, peptide nucleic acids (PNA's) and 2′-5′linkages. Another modification to oligonucleotides, usually fordiagnostic and research applications, is labeling with non-isotopiclabels, e.g., fluorescein, biotin, digoxigenin, alkaline phosphatase, orother reporter molecules.

A variety of modified phosphorus-containing linkages have been studiedas replacements for the natural, readily cleaved phosphodiester linkagein oligonucleotides. In general, most of them (such as thephosphorothioate, phosphoramidates, phosphonates andphosphorodithioates) result in oligonucleotides with reduced binding tocomplementary targets and decreased hybrid stability. At least one dozenphosphorothioate oligonucleotides and derivatives are presently beingused as antisense agents in human clinical trials for the treatment ofvarious disease states. The antisense drug Vitravin™, for use to treatcytomegalovirus (CMV) retinitis in humans, has been approved byregulatory agencies and is comedically marketed.

The structure and stability of chemically modified nucleic acids is ofgreat importance to the design of antisense oligonucleotides. Over thelast ten years, a variety of synthetic modifications have been proposedto increase nuclease resistance, or to enhance the affinity of theantisense strand for its target mRNA (Crooke et al., Med. Res. Rev.,1996, 16, 319-344; De Mesmaeker et al., Acc. Chem. Res., 1995, 28,366-374).

RNA exists in what has been termed “A Form” geometry, while DNA existsin “B Form” geometry. In general, RNA:RNA duplexes are more stable, orhave higher melting temperatures (Tm) than DNA:DNA duplexes (Sanger etal., Principles of Nucleic Acid Structure, 1984, Springer-Verlag; NewYork, N.Y.; Lesnik et al., Biochemistry, 1995, 34, 10807-10815; Conte etal., Nucleic Acids Res., 1997, 25, 2627-2634). The increased stabilityof RNA has been attributed to several structural features, most notablythe improved base stacking interactions that result from an A-formgeometry (Searle et al., Nucleic Acids Res., 1993, 21, 2051-2056). Thepresence of a hydroxyl group in the 2′-pentofuranosyl (i.e., 2′-sugar)position in RNA is believed to bias the sugar toward a C3′ endo pucker(also known as a Northern pucker), which causes the duplex to favor theA-form geometry. On the other hand, 2′-deoxy nucleic acids (those having2′-deoxy-erythro-pentofuranosyl nucleotides) prefer a C2′ endo sugarpucker (also known as Southern pucker), which is thought to impart aless stable B-form geometry (Sanger, W. (1984) Principles of NucleicAcid Structure, Springer-Verlag, New York, N.Y.). In addition, the 2′hydroxyl groups of RNA can form a network of water mediated hydrogenbonds that help stabilize the RNA duplex (Egli et al., Biochemistry,1996, 35, 8489-8494).

DNA:RNA hybrid duplexes are usually less stable than pure RNA:RNAduplexes, and depending on their sequence may be either more or lessstable than DNA:DNA duplexes (Searle et al., Nucleic Acids Res., 1993,21, 2051-2056). The structure of a hybrid duplex is intermediate betweenA- and B-form geometries, which may result in poor stacking interactions(Lane et al., Eur. J. Biochem., 1993, 215, 297-306; Fedoroffet al., J.Mol. Biol., 1993, 233, 509-523; Gonzalez et al., Biochemistry, 1995, 34,4969-4982; Hortonetal., J. Mol. Biol., 1996, 264, 521-533). Thestability of a DNA:RNA hybrid is central to antisense therapies as themechanism requires the binding of a modified DNA strand to a mRNAstrand. To effectively inhibit the mRNA, the antisense DNA should have avery high binding affinity with the mRNA. Otherwise the desiredinteraction between the DNA and target mRNA strand will occurinfrequently, thereby decreasing the efficacy of the antisenseoligonucleotide.

One synthetic 2′-modification that imparts increased nuclease resistanceand a very high binding affinity to nucleotides is the 2′-methoxyethoxy(MOE, 2′-OCH₂CH₂OCH₃) side chain (Baker et al., J. Biol. Chem., 1997,272, 11944-12000; Freier et al., Nucleic Acids Res., 1997, 25,4429-4443). One of the immediate advantages of the MOE substitution isthe improvement in binding affinity, which is greater than many similar2′ modifications such as O-methyl, O-propyl, and O-aminopropyl (Freierand Altmann, Nucleic Acids Research, (1997) 25:4429-4443).2′-O-Methoxyethyl-substituted compounds also have been shown to beantisense inhibitors of gene expression with promising features for invivo use (Martin, P., Helv. Chim. Acta, 1995, 78, 486-504; Altmann etal., Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Soc. Trans.,1996, 24, 630-637; and Altmann et al., Nucleosides Nucleotides, 1997,16, 917-926). Such compounds typically display improved RNA affinity andhigher nuclease resistance relative to DNA. Chimeric oligonucleotideswith 2′-O-methoxyethyl-ribonucleoside wings and a centralDNA-phosphorothioate window also have been shown to effectively reducethe growth of tumors in animal models at low doses. MOE substitutedoligonucleotides have shown outstanding promise as antisense agents inseveral disease states. One such MOE substituted oligonucleotide ispresently being investigated in clinical trials for the treatment of CMVretinitis.

Recently Damha et. al., published two paper describing certainoligonucleotides that utilized arabino-pentofuranosyl nucleotides asbuilding blocks (Damha et. al., J.A.C.S., 1998, 120, 12976-12977 andDamha et. al., Bioconjugate Chem., 1999, 10, 299-305). Thearabino-pentofuranosyl oligonucleotides, i.e., arabinonucleic acids,described by Damha et. al., utilized either arabinose or2′-deoxy-2′-fluoro arabinose as the sugar unit of their respectivenucleotides. In one of the two arabinonucleic acids described, all ofthe nucleotides of the nucleic acid were arabinose and in the other, allof the nucleotides were 2′-deoxy-2′-fluoro arabinose. In both of thesenucleic acids, the nucleotides were joined via phosphodiester linkages.These authors were able to show that the 2′-fluoro arabino-containingoligonucleotides when bound to RNA activated cleavage of the RNA by E.coli and HIV-RT RNase H. The authors further noted that while the twoarabinonucleic acids they described were more stable to serum andcellular nucleases than DNA they were less stable than normalphosphorothioate deoxyoligonucleotides.

Although the known modifications to oligonucleotides have contributed tothe development of oligonuclotides for various uses, including use indiagnostics, therapeutics and as research reagents, there still exists aneed in the art for further oligonucleotides having enhanced hybridbinding affinity and/or increased nuclease resistance and that can takeadvantage of the RNase H termination mechanism.

SUMMARY OF THE INVENTION

In one aspect, the present invention is directed to oligonucleotideshaving multiple properties. One of these properties is the ability toform a double stranded structure with an RNA and elicit RNase H cleavageof the RNA. Further properties of the oligonucleotides include havingimproved binding affinity and nuclease resistance. The oligonucleotidesof the invention comprise oligonucleotide formed from a plurality ofnucleotides. A first portion of the nucleotides are joined together in acontiguous sequence with each nucleotide of this portion selected as anucleotide that has B-form conformational geometry when joined in acontiguous sequence with other nucleotides. Included in this firstportion of nucleotides are ribonucleotides or arabino nucleotides. Theoligonucleotides include a further portion of nucleotides that arejoined together in at least one contiguous sequence. Each of thesefurther nucleotides are selected as ribonucleotides that have A-formconformational geometry when joined in a contiguous sequence.

In preferred embodiments of the invention, each of the nucleotides ofthe first portion of nucleotides, independently, are selected to be2′-SCH₃ ribonucleotides, 2′-NH₂ ribonucleotides, 2′-NH(C₁-C₂alkyl)ribonucleotides, 2′-N(C₁-C₂ alkyl)₂ ribonucleotides, 2′-CF₃ribonucleotides, 2′=CH₂ ribonucleotides, 2′=CHF ribonucleotides, 2′=CF₂ribonucleotides, 2′-CH₃ ribonucleotides, 2′-C₂H₅ ribonucleotides,2′-CH═CH₂ ribonucleotides or 2′-C≡CH ribonucleotides. These are joinedtogether in a contiguous sequence by phosphate, phosphorothioate,phosphorodithioate or boranophosphate linkages.

In a further preferred embodiment of the invention, each of thenucleotides of said further portion of nucleotides, independently, areselected to be 2′-fluoro nucleotides or nucleotides having a2′-substituenthaving the formula I or II:

wherein

-   -   E is C₁-C₁₀ alkyl, N(Q₁)(Q₂) or N═C(Q₁)(Q₂);

each Q₁ and Q₂ is, independently, H, C₁-C₁₀ alkyl, dialkylaminoalkyl, anitrogen protecting group, a tethered or untethered conjugate group, alinker to a solid support, or Q₁ and Q₂, together, are joined in anitrogen protecting group or a ring structure that can include at leastone additional heteroatom selected from N and O;

-   -   R₃ is OX, SX, or N(X)₂;    -   each X is, independently, H, C₁-C₈ alkyl, C₁-C₈ haloalkyl,        C(═NH)N(H)Z, C(═O)N(H)Z or OC(═O)N(H)Z;    -   Z is H or C₁-C₈ alkyl;

L₁, L₂ and L₃ comprise a ring system having from about 4 to about 7carbon atoms or having from about 3 to about 6 carbon atoms and 1 or 2hetero atoms wherein said hetero atoms are selected from oxygen,nitrogen and sulfur and wherein said ring system is aliphatic,unsaturated aliphatic, aromatic, or saturated or unsaturatedheterocyclic;

Y is alkyl or haloalkyl having 1 to about 10 carbon atoms, alkenylhaving 2 to about 10 carbon atoms, alkynyl having 2 to about 10 carbonatoms, aryl having 6 to about 14 carbon atoms, N(Q₁)(Q₂), O(Q₁), halo,S(Q₁), or CN;

-   -   each q₁ is, independently, from 2 to 10;    -   each q₂ is, independently, 0 or 1;    -   m is 0, 1 or 2;    -   p is from 1 to 10; and    -   q₃ is from 1 to 10 with the proviso that when p is 0, q₃ is        greater than 1.

A more preferred group for use as the further portion of nucleotides are2′-F ribonucleotides, 2′-O—(C₁-C₆ alkyl)ribonucleotides, or 2′-O—(C₁-C₆substituted alkyl)ribonucleotides wherein the substitution is C₁-C₆ether, C₁-C₆ thioether, amino,amino(C₁-C₆ alkyl) or amino(C₁-C₆ alkyl)₂.These nucleotides are joined together in sequence by 3′-5′phosphodiester, 2′-5′ phosphodiester, phosphorothioate, Spphosphorothioate, Rp phosphorothioate, phosphorodithioate,3′-deoxy-3′-amino phosphoroarnidate, 3′-methylenephosphonate,methylene(methylimino), dimethylhydazino, amide 3 (i.e.,(3′)—CH₂—NH—C(O)—(5′)), amide 4 (i.e., (3′)—CH₂—C(O)—NH—(5′) orboranophosphate linkages.

In one preferred embodiment of the invention, at least two of thenucleotides of the further portion of nucleotides are joined together ina contiguous sequence that is position 3′ to the contiguous sequence ofthe first portion of nucleotides. In an additional preferred embodimentof the invention, at least two of the further portion of nucleotides arejoined together in a continuous sequence that is position 5′ to thecontinuous sequence of the first portion of nucleotides.

In a further preferred embodiment of the invention, at least two of thenucleotides of the further portion of nucleotides are joined together ina continuous sequence that is position 3′ to the continuous sequence ofthe first portion of nucleotides and at least two of the further portionof nucleotides are joined together in a continuous sequence that isposition 5′ to the continuous sequence of the first portion ofnucleotides.

A first preferred group of nucleotides for use as the first portion ofnucleotides include 2′-SCH₃ ribonucleotides, 2′-NH2 ribonucleotides,2′-NH(C₁-C₂ alkyl)ribonucleotides, 2′-N(C₁-C₂ alkyl)₂ ribonucleotides,2′=CH₂ ribonucleotides, 2′-CH₃ ribonucleotides, 2′-C₂H₅ ribonucleotides,2′-CH═CH₂ ribonucleotides and 2′-C≡CH ribonucleotides. A more preferredgroup include 2′-SCH₃ ribonucleotides, 2′-NH₂ ribonucleotides,2′-NH(C₁-C₂ alkyl)ribonucleotides, 2′-N(C₁-C₂ alkyl)₂ ribonucleotidesand 2′-CH₃ ribonucleotides. A further preferred group include 2′-SCH₃ribonucleotides, 2′-NH₂ribonucleotides and 2′-CH₃ribonucleotides.Particularly preferred are is 2′-SCH₃ ribonucleotides.

A further group of nucleotides that are preferred of use as thenucleotides of the first portion of the oligonucleotides of theinventions are 2′-CN arabino nucleotides, 2′-F arabino nucleotides,2′-Cl arabino nucleotides, 2′-Br arabino nucleotides, 2′-N₃ arabinonucleotides, 2′-OH arabino nucleotides, 2′-O—CH₃ arabino nucleotides and2′-dehydro-2′-CH₃ arabino nucleotides. A more preferred group include2′-F arabino nucleotides, 2′-OH arabino nucleotides and 2′-O—CH₃ arabinonucleotides. A further preferred group include 2′-F arabino nucleotidesand 2′-OH arabino nucleotides. Particularly preferred are 2′-F arabinonucleotides.

Particularly preferred oligonucleotides of the invention includeselecting the nucleotides of the first portion of nucleotides to be2′-SCH₃ ribonucleotides, 2′-NH₂ ribonucleotides, 2′-NH(C₁-C₂alkyl)ribonucleotides, 2′-N(C₁-C₂ alkyl)₂ ribonucleotides, 2′-CH₃ribonucleotides, 2′-CH═CH₂ ribonucleotides or 2′-C≡CH ribonucleotidesand selecting the nucleotides of the further portion of nucleotides tobe 2′-F ribonucleotides, 2′-O—(C₁-C₆ alkyl)ribonucleotides or2′-O—(C₁-C₆ substituted alkyl)ribonucleotides wherein the substitutionis C₁-C₆ ether, C₁-C₆ thioether, amino, amino(C₁-C₆ alkyl) or amino(C₁-C₆ alkyl)₂.

Further preferred oligonucleotides of the invention include selectingthe nucleotides of said first portion of nucleotides to be 2′-CN arabinonucleotides, 2′-F arabino nucleotides, 2′-Cl arabino nucleotides, 2′-Brarabino nucleotides, 2′-N₃ arabino nucleotides, 2′-OH arabinonucleotides, 2′-O—CH₃ arabino nucleotides or 2′-dehydro-2′-CH₃ arabinonucleotides and selecting the nucleotides of the further portion ofnucleotides to be 2′-F ribonucleotides, 2′-O—(C₁-C₆alkyl)ribonucleotides or 2′-O—(C₁-C₆ substituted alkyl)ribonucleotideswherein the substitution is C₁-C₆ ether, C₁-C₆ thioether, amino,amino(C₁-C₆ alkyl) or amino(C₁-C₆ alkyl)₂.

Particularly preferred are oligonucleotide of the invention where eachnucleotide of the first portion of nucleotides is a 2′-F arabinonucleotides or a 2′-OH arabino nucleotides and each nucleotide of thefurther portion of nucleotides is a 2′-O—(C₁-C₆ substitutedalkyl)ribonucleotide wherein the substitution is C₁-C₆ ether, C₁-C₆thioether, amino, amino(C₁-C₆ alkyl) or amino(C₁-C₆ alkyl)₂.

In further preferred oligonucleotides of the invention the furtherportion of the plurality of nucleotides comprise at least twonucleotides joined together in a contiguous sequence that is position atthe 3′ terminus end of the oligonucleotide. In an additional preferredoligonucleotide of the invention the further portion of said pluralityof nucleotides comprise at least two nucleotides joined together in acontiguous sequence that is position at the 5′ terminus end of theoligonucleotide. In even further preferred oligonucleotides of theinvention the further portion of the plurality of nucleotides compriseat least two nucleotides joined together in a contiguous sequence thatis position at the 3′ terminus end of the oligonucleotide and at leasttwo nucleotides joined together in a contiguous sequence that isposition at the 5′ terminus end of the oligonucleotide. Preferredlinkages for joining these nucleotides together in an oligonucleotide ofthe invention include 2′-5′ phosphodiester linkages,3′-methylenephosphonate linkages, Sp phosphorothioate linkages,methylene(methylimino)linkages, dimethyhydrazino linkages,3′-deoxy-3′-amino phosphoroamidate linkages, amide 3 linkages or amide 4linkages. Particularly preferred joining linkages are 2′-5′phosphodiester linkages, 3′-methylenephosphonate linkages, Spphosphorothioate linkages or methylene(methylimino) linkages.

In further preferred oligonucleotides of the invention, nucleotides foruse in the further portion of nucleotides comprises 2′-alkylaminosubstituted nucleotides located at the 3′ terminus, the 5′ terminus orboth the 3′ and 5′ terminus of the oligonucleotide. Particularlypreferred are 2′-O-alkylamines such as 2′-O-ethylamine and2′-O-propylamine.

Further oligonucleotides of the invention comprise oligonucleotides madeup of a plurality of linked nucleotides at least two of which comprisenucleotides that are not 2′-deoxy-erthro-pentofuranosyl nucleotides andthat have a C2′ endo type pucker or an O4′ endo type pucker and that arejoined together in a contiguous sequence and other nucleotidescomprising nucleotides that have a C3′ endo type pucker. Preferred areoligonucleotides having the C3′ endo type pucker nucleotides joinedtogether in a contiguous sequence that is positioned 3′ to thecontiguous sequence of the nucleotides having the C2′ endo type puckeror O4′ endo type pucker. Further preferred oligonucleotides areoligonucleotides wherein the nucleotides having the C3′ endo type puckerare joined together in a contiguous sequence that is positioned 5′ tothe contiguous sequence of having the C2′ endo type pucker or O4′ endotype pucker. Additional preferred oligonucleotide are oligonucleotideswhere a portion of the nucleotides having the C3′ endo type pucker arejoined together in a contiguous sequence that is positioned 3′ to thecontiguous sequence of nucleotides having the C2′ endo type pucker orO4′ endo type pucker and a further portion of nucleotides having the C3′endo type pucker are joined together in a contiguous sequence that ispositioned 5′ to the contiguous sequence of nucleotides having the C2′endo type pucker or O4′ endo type pucker.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a preferred group of nucleotide fragments for use inthe B-form portion (the C2′ endo/O4′ endo portion) of oligonucleotidesof the invention.

FIG. 2 illustrates a preferred group of nucleotide fragments for use inthe A-form portion (the C3′ endo portion) of oligonucleotides of theinvention.

FIG. 3 illustrates a preferred group of nucleotide fragments for use inA-form portions at the 3′ terminus of oligonucleotides of the invention.

FIG. 4 is a plot of the percentage of full length oligonucleotideremaining intact in plasma one hour following administration of an i.v.bolus of 5 mg/kg oligonucleotide.

FIG. 5 is a plot of the percentage of full length oligonucleotideremaining intact in tissue 24 hours following administration of an i.v.bolus of 5 mg/kg oligonucleotide.

FIG. 6 shows CGE traces of test oligonucleotides and a standardphosphorothioate oligonucleotide in both mouse liver samples and mousekidney samples after 24 hours.

FIG. 7 shows a graph of the effect of the oligonucleotides of thepresent invention on c-raf expression (compared to control) in bENDcells.

FIGS. 8 and 9 shows bar graphs as percent control normalized for theG3PDH signal eighteen hours after treatment with specified compounds.

DESCRIPTION OF PREFERRED EMBODIMENTS

In one aspect, the present invention is directed to noveloligonucleotides that have certain desirable properties that contributeto increases in binding affinity and/or nuclease resistance, coupledwith the ability to serve as substrates for RNase H.

The oligonucleotide of the invention are formed from a plurality ofnucleotides that are joined together via internucleotide linkages. Whilejoined together as a unit in the oligonucleotide, the individualnucleotides of oligonucleotides are of several types. Each of thesetypes contribute unique properties to the oligonucleotide. A first typeof nucleotides are joined together in a continuous sequence that forms afirst portion of the oligonucleotide. The remaining nucleotides are ofat least one further type and are located in one or more remainingportions or locations within the oligonucleotide. Thus, theoligonucleotides of the invention include a nucleotide portion thatcontributes one set of attributes and a further portion (or portions)that contributes another set of attributes.

One attribute that is desirable is eliciting RNase H activity. To elicitRNase H activity, a portion of the oligonucleotides of the invention isselected to have B-form like conformational geometry. The nucleotidesfor this B-form portion are selected to specifically includeribo-pentofuranosyl and arabino-pentofuranosyl nucleotides.2′-Deoxy-erythro-pentfuranosyl nucleotides also have B-form geometry andelicit RNase H activity. While not specifically excluded, if2′-deoxy-erythro-pentfuranosyl nucleotides are included in the B-formportion of an oligonucleotide of the invention, such2′-deoxy-erythro-pentfuranosyl nucleotides preferably does notconstitute the totality of the nucleotides of that B-form portion of theoligonucleotide, but should be used in conjunction with ribonucleotidesor an arabino nucleotides. As used herein, B-form geometry is inclusiveof both C2′-endo and O4′-endo pucker, and the ribo and arabinonucleotides selected for inclusion in the oligonucleotide B-form portionare selected to be those nucleotides having C2′-endo conformation orthose nucleotides having O4′-endo conformation. This is consistent withBerger, et. al., Nucleic Acids Research, 1998, 26, 2473-2480, whopointed out that in considering the furanose conformations in whichnucleosides and nucleotides reside, B-form consideration should also begiven to a O4′-endo pucker contribution.

A-form nucleotides are nucleotides that exhibit C3′-endo pucker, alsoknown as north, or northern, pucker. In addition to the B-formnucleotides noted above, the A-form nucleotides can be C3′-endo puckernucleotides or can be nucleotides that are located at the 3′ terminus,at the 5′ terminus, or at both the 3′ and the 5′ terminus of theoligonucleotide. Alternatively, A-form nucleotides can exist both in aC3′-endo pucker and be located at the ends, or termini, of theoligonucleotide. In selecting nucleotides that have C3′-endo pucker orin selecting nucleotides to reside at the 3′ or 5′ ends of theoligonucleotide, consideration is given to binding affinity and nucleaseresistance properties that such nucleotides need to impart to theresulting the oligonucleotide.

Nucleotides selected to reside at the 3′ or 5′ termini ofoligonucleotides of the invention are selected to impart nucleaseresistance to the oligonucleotide. This nuclease resistance can also beachieved via several mechanisms, including modifications of the sugarportions of the nucleotide units of the oligonucleotides, modificationof the internucleotide linkages or both modification of the sugar andthe internucleotide linkage.

A particularly useful group of nucleotides for use in increasingnuclease resistance at the termini of oligonucleotides are those having2′-O-alkylamino groups thereon. The amino groups of such nucleotides canbe groups that are protonated at physiological pH. These include amines,monoalkyl substituted amines, dialkyl substituted amines andheterocyclic amines such as imidazole. Particularly useful are the loweralkyl amines including 2′-O-ethylamine and 2′-O-propylamine. SuchO-alkylamines can also be included on the 3′ position of the 3′ terminusnucleotide. Thus the 3′ terminus nucleotide could include both a 2′ anda 3′-O-alkylamino substituent.

In selecting for nuclease resistance, it is important not to detractfrom binding affinity. Certain phosphorus based linkage have been shownto increase nuclease resistance. The above described phosphorothioatelinkage increase nuclease resistance, however, it also causes loss ofbinding affinity. Thus, generally for use in this invention, ifphosphorothioate internucleotide linkage are used, other modificationwill be made to nucleotide units that increase binding affinity tocompensate for the decreased affinity contribute by the phosphorothioatelinkages.

Other phosphorus based linkages having increase nuclease resistance thatdo not detract from binding affinity include 3′-methylene phosphonatesand 3′-deoxy-3′-amino-phosphoroamidate linkages. A further class oflinkages that contribute nuclease resistance but do not detract frombinding affinity are non-phosphate in nature. Preferred among these aremethylene(methylimino) linkages, dimethylhydraxino linkages, and amine 3and amide 4 linkages as described (Freier and Altmann, Nucleic AcidResearch, 1997, 25, 4429-4443).

There are a number of potential items to consider when designingoligonucleotides having improved binding affinities. It appears that oneeffective approach to constructing modified oligonucleotides with veryhigh RNA binding affinity is the combination of two or more differenttypes of modifications, each of which contributes favorably to variousfactors that might be important for binding affinity.

Freier and Altmann, Nucleic Acids Research, (1997) 25:4429-4443,recently published a study on the influence of structural modificationsof oligonucleotides on the stability of their duplexes with target RNA.In this study, the authors reviewed a series of oligonucleotidescontaining more than 200 different modifications that had beensynthesized and assessed for their hybridization affinity and T_(m).Sugar modifications studied included substitutions on the 2′-position ofthe sugar, 3′-substitution, replacement of the 4′-oxygen, the use ofbicyclic sugars, and four member ring replacements. Several nucleobasemodifications were also studied including substitutions at the 5, or 6position of thymine, modifications of pyrimidine heterocycle andmodifications of the purine heterocycle. Numerous backbone modificationswere also investigated including backbones bearing phosphorus, backbonesthat did not bear a phosphorus atom, and backbones that were neutral.

Four general approaches might be used to improve hybridization ofoligonucleotides to RNA targets. These include: preorganization of thesugars and phosphates of the oligodeoxynucleotide strand intoconformations favorable for hybrid formation, improving stacking ofnucleobases by the addition of polarizable groups to the heterocyclebases of the nucleotides of the oligonucleotide, increasing the numberof H-bonds available for A-U pairing, and neutralization of backbonecharge to facilitate removing undesirable repulsive interactions. Wehave found that by utilizing the first of these, preorganization of thesugars and phosphates of the oligodeoxynucleotide strand intoconformations favorable for hybrid formation, to be a preferred methodto achieve improve binding affinity. It can further be used incombination with the other three approaches.

Sugars in DNA:RNA hybrid duplexes frequently adopt a C3′ endoconformation. Thus modifications that shift the conformationalequilibrium of the sugar moieties in the single strand toward thisconformation should preorganize the antisense strand for binding to RNA.Of the several sugar modifications that have been reported and studiedin the literature, the incorporation of electronegative substituentssuch as 2′-fluoro or 2′-alkoxy shift the sugar conformation towards the3′ endo (northern) pucker conformation. This preorganizes anoligonucleotide that incorporates such modifications to have an A-formconformational geometry. This A-form conformation results in increasedbinding affinity of the oligonucleotide to a target RNA strand.

Representative 2′-substituent groups amenable to the present inventionthat give A-form conformational properties to the nucleotides include2′-O-alkyl, 2′-O-substituted alkyl and 2′-fluoro substituent groups.Preferred for the substituent groups are various alkyl and aryl ethersand thioethers, amines and monoalkyl and dialkyl substituted amines. Aparticular preferred group include those having the formula I or II:

wherein

-   -   E is C₁-C₁₀ alkyl, N(Q₁)(Q₂) or N═C(Q₁)(Q2);

each Q₁ and Q₂ is, independently, H, C₁-C₁₀ alkyl, dialkylaminoalkyl, anitrogen protecting group, a tethered or untethered conjugate group, alinker to a solid support, or Q₁ and Q₂, together, are joined in anitrogen protecting group or a ring structure that can include at leastone additional heteroatom selected from N and O;

-   -   R₃ is OX, SX, or N(X)₂;    -   each X is, independently, H, C₁-C₈ alkyl, C₁-C₈ haloalkyl,        C(═NH)N(H)Z, C(═O)N(H)Z or OC(═O)N(H)Z;    -   Z is H or C₁-C8 alkyl;

L₁, L₂ and L₃ comprise a ring system having from about 4 to about 7carbon atoms or having from about 3 to about 6 carbon atoms and 1 or 2hetero atoms wherein said hetero atoms are selected from oxygen,nitrogen and sulfur and wherein said ring system is aliphatic,unsaturated aliphatic, aromatic, or saturated or unsaturatedheterocyclic;

Y is alkyl or haloalkyl having 1 to about 10 carbon atoms, alkenylhaving 2 to about 10 carbon atoms, alkynyl having 2 to about 10 carbonatoms, aryl having 6 to about 14 carbon atoms, N(Q₁)(Q₂), O(Q₁), halo,S(Q₁), or CN;

-   -   each q₁ is, independently, from 2 to 10;    -   each q₂ is, independently, 0 or 1;    -   m is 0, 1 or 2;    -   p is from 1 to 10; and    -   q₃ is from 1 to 10 with the proviso that when p is 0, q₃ is        greater than 1.

The above 2′-substituents confer a 3′-endo pucker to the sugar wherethey are incorporated. This pucker conformation further assists inincreasing the Tm of the oligonucleotide with its target.

The high binding affinity resulting from 2′ substitution has beenpartially attributed to the 2′ substitution causing a C3′ endo sugarpucker which in turn may give the oligomer a favorable A-form likegeometry. This is a reasonable hypothesis since substitution at the 2′position by a variety of electronegative groups (such as fluoro andO-alkyl chains) has been demonstrated to cause C3′ endo sugar puckering(De Mesmaeker et al., Acc. Chem. Res., 1995, 28, 366-374; Lesnik et al.,Biochemistry, 1993, 32, 7832-7838).

In addition, for 2′-substituents containing an ethylene glycol motif, agauche interaction between the oxygen atoms around the O—C—C—O torsionof the side chain may have a stabilizing effect on the duplex (Freier etal., Nucleic Acids Research, (1997) 25:4429-4442). Such gaucheinteractions have been observed experimentally for a number of years(Wolfe et al., Acc. Chem. Res., 1972, 5, 102; Abe et al., J. Am. Chem.Soc., 1976, 98, 468). This gauche effect may result in a configurationof the side chain that is favorable for duplex formation. The exactnature of this stabilizing configuration has not yet been explained.While we do not want to be bound by theory, it may be that holding theO—C—C—O torsion in a single gauche configuration, rather than a morerandom distribution seen in an alkyl side chain, provides an entropicadvantage for duplex formation.

To better understand the higher RNA affinity of 2′-O-methoxyethylsubstituted RNA and to examine the conformational properties of the2′-O-methoxyethyl substituent, a self-complementary dodecameroligonucleotide 2′-O-MOE r(CGCGAAUUCGCG) SEQ ID NO: 1 was synthesized,crystallized and its structure at a resolution of 1.7 Ångstrom wasdetermined. The crystallization conditions used were 2 mMoligonucleotide, 50 mM Na Hepes pH 6.2-7.5, 10.50 mM MgCl₂, 15% PEG 400.The crystal data showed: space group C2, cell constants a=41.2 Å, b=34.4Å, c=46.6 Å, β=92.4°. The resolution was 1.7 Å at −170° C. The currentR=factor was 20% (R_(free) 26%).

This crystal structure is believed to be the first crystal structure ofa fully modified RNA oligonucleotide analogue. The duplex adopts anoverall A-form conformation and all modified sugars display C3′-endopucker. In most of the 2′-O-substituents, the torsion angle around theA′-B′ as depicted in Structure II below, of the ethylene glycol linkerhas a gauche conformation. For 2′-O-MOE, A′ and B′ of Structure II beloware methylene moieties of the ethyl portion of the MOE and R′ is themethoxy portion.

In the crystal, the 2′-O-MOE RNA duplex adopts a general orientationsuch that the crystallographic 2-fold rotation axis does not coincidewith the molecular 2-fold rotation axis. The duplex adopts the expectedA-type geometry and all of the 24 2′-O-MOE substituents were visible inthe electron density maps at full resolution. The electron density mapsas well as the temperature factors of substituent atoms indicateflexibility of the 2′-O-MOE substituent in some cases.

Most of the 2-O-MOE substituents display a gauche conformation aroundthe C—C bond of the ethyl linker. However, in two cases, a transconformation around the C—C bond is observed. The lattice interactionsin the crystal include packing of duplexes against each other via theirminor grooves. Therefore, for some residues, the conformation of the2′-O-substituent is affected by contacts to an adjacent duplex. Ingeneral, variations in the conformation of the substituents (e.g. g⁺ org⁻ around the C—C bonds) create a range of interactions betweensubstituents, both inter-strand, across the minor groove, andintra-strand. At one location, atoms of substituents from two residuesare in van der Waals contact across the minor groove. Similarly, a closecontact occurs between atoms of substituents from two adjacentintra-strand residues.

Previously determined crystal structures of A-DNA duplexes were forthose that incorporated isolated 2′-O-methyl T residues. In the crystalstructure noted above for the 2′-O-MOE substituents, a conservedhydration pattern has been observed for the 2′-O-MOE residues. A singlewater molecule is seen located between O2′, O3′ and the methoxy oxygenatom of the substituent, forming contacts to all three of between 2.9and 3.4 Å. In addition, oxygen atoms of substituents are involved inseveral other hydrogen bonding contacts. For example, the methoxy oxygenatom of a particular 2′-O-substituent forms a hydrogen bond to N3 of anadenosine from the opposite strand via a bridging water molecule.

In several cases a water molecule is trapped between the oxygen atomsO2′, O3′ and OC′ of modified nucleosides. 2′-O-MOE substituents withtrans conformation around the C—C bond of the ethylene glycol linker areassociated with close contacts between OC′ and N2 of a guanosine fromthe opposite strand, and, water-mediated, between OC′ and N3(G). Whencombined with the available thermodynamic data for duplexes containing2′-O-MOE modified strands, this crystal structure allows for furtherdetailed structure-stability analysis of other antisense modifications.

In extending the crystallographic structure studies, molecular modelingexperiments were performed to study further enhanced binding affinity ofoligonucleotides having 2′-O-modifications of the invention. Thecomputer simulations were conducted on compounds of SEQ ID NO: 1, above,having 2′-O-modifications of the invention located at each of thenucleoside of the oligonucleotide. The simulations were performed withthe oligonucleotide in aqueous solution using the AMBER force fieldmethod (Cornell et al., J. Am. Chem. Soc., 1995,117, 5179-5197)(modelingsoftware package from UCSF, San Francisco, Calif.). The calculationswere performed on an Indigo2 SGI machine (Silicon Graphics, MountainView, Calif.).

Further 2′-O-modifications of the inventions include those having a ringstructure that incorporates a two atom portion corresponding to the A′and B′ atoms of Structure II. The ring structure is attached at the 2′position of a sugar moiety of one or more nucleosides that areincorporated into an oligonucleotide. The 2′-oxygen of the nucleosidelinks to a carbon atom corresponding to the A′ atom of Structure II.These ring structures can be aliphatic, unsaturated aliphatic, aromaticor heterocyclic. A further atom of the ring (corresponding to the B′atom of Structure II), bears a further oxygen atom, or a sulfur ornitrogen atom. This oxygen, sulfur or nitrogen atom is bonded to one ormore hydrogen atoms, alkyl moieties, or haloalkyl moieties, or is partof a further chemical moiety such as a ureido, carbamate, amide oramidine moiety. The remainder of the ring structure restricts rotationabout the bond joining these two ring atoms. This assists in positioningthe “further oxygen, sulfur or nitrogen atom” (part of the R position asdescribed above) such that the further atom can be located in closeproximity to the 3′-oxygen atom (O3′) of the nucleoside.

The ring structure can be further modified with a group useful formodifying the hydrophilic and hydrophobic properties of the ring towhich it is attached and thus the properties of an oligonucleotide thatincludes the 2′-O-modifications of the invention. Further groups can beselected as groups capable of assuming a charged structure, e.g. anamine. This is particularly useful in modifying the overall charge of anoligonucleotide that includes a 2′-O-modifications of the invention.When an oligonucleotide is linked by charged phosphate groups, e.g.phosphorothioate or phosphodiester linkages, location of a counter ionon the 2′-O-modification, e.g. an amine functionality, locallynaturalizes the charge in the local environment of the nucleotidebearing the 2′-O-modification. Such neutralization of charge willmodulate uptake, cell localization and other pharmacokinetic andpharmacodynamic effects of the oligonucleotide.

Preferred ring structures of the invention for inclusion as a 2′-Omodification include cyclohexyl, cyclopentyl and phenyl rings as well asheterocyclic rings having spacial footprints similar to cyclohexyl,cyclopentyl and phenyl rings. Particularly preferred 2′-O-substituentgroups of the invention are listed below including an abbreviation foreach:

-   -   2′-O-(trans 2-methoxy cyclohexyl)—2′-O-(TMCHL)    -   2′-O-(trans 2-methoxy cyclopentyl)—2′-O-(TMCPL)    -   2′-O-(trans 2-ureido cyclohexyl)—2′-O-(TUCHL)    -   2′-O-(trans 2-methoxyphenyl)—2′-O-(2MP)

Structural details for duplexes incorporating such 2-O-substituents wereanalyzed using the described AMBER force field program on the Indigo2SGI machine. The simulated structure maintained a stable A-form geometrythroughout the duration of the simulation. The presence of the 2′substitutions locked the sugars in the C3′-endo conformation.

The simulation for the TMCHL modification revealed that the 2′-O-(TMCHL)side chains have a direct interaction with water molecules solvating theduplex. The oxygen atoms in the 2-O-(TMCHL) side chain are capable offorming a water-mediated interaction with the 3′ oxygen of the phosphatebackbone. The presence of the two oxygen atoms in the 2′-O-(TMCHL) sidechain gives rise to favorable gauche interactions. The barrier forrotation around the O—C—C—O torsion is made even larger by this novelmodification. The preferential preorganization in an A-type geometryincreases the binding affinity of the 2′-O-(TMCHL) to the target RNA.The locked side chain conformation in the 2′-O-(TMCHL) group created amore favorable pocket for binding water molecules. The presence of thesewater molecules played a key role in holding the side chains in thepreferable gauche conformation. While not wishing to be bound by theory,the bulk of the substituent, the diequatorial orientation of thesubstituents in the cyclohexane ring, the water of hydration and thepotential for trapping of metal ions in the conformation generated willadditionally contribute to improved binding affinity and nucleaseresistance of oligonucleotides incorporating nucleosides having this2′-O-modification.

As described for the TMCHL modification above, identical computersimulations of the 2′-O-(TMCPL), the 2′-O-(2MP) and 2′-O-(TUCHL)modified oligonucleotides in aqueous solution also illustrate thatstable A-form geometry will be maintained throughout the duration of thesimulation. The presence of the 2′ substitution will lock the sugars inthe C3′-endo conformation and the side chains will have directinteraction with water molecules solvating the duplex. The oxygen atomsin the respective side chains are capable of forming a water-mediatedinteraction with the 3′ oxygen of the phosphate backbone. The presenceof the two oxygen atoms in the respective side chains give rise to thefavorable gauche interactions. The barrier for rotation around therespective O—C—C—O torsions will be made even larger by respectivemodification. The preferential preorganization in A-type geometry willincrease the binding affinity of the respective 2′-O-modifiedoligonucleotides to the target RNA. The locked side chain conformationin the respective modifications will create a more favorable pocket forbinding watermolecules. The presence of these water molecules plays akey role in holding the side chains in the preferable gaucheconformation. The bulk of the substituent, the diequatorial orientationof the substituents in their respective rings, the water of hydrationand the potential trapping of metal ions in the conformation generatedwill all contribute to improved binding affinity and nuclease resistanceof oligonucleotides incorporating nucleosides having these respective2′-O-modification.

Preferred for use as the B-form nucleotides for eliciting RNase H areribonucleotides having 2-deoxy-2′-S-methyl, 2′-deoxy-2′-methyl,2′-deoxy-2′-amino, 2′-deoxy-2′-mono or dialkyl substituted amino,2′-deoxy-2′-fluoromethyl, 2′-deoxy-2′-difluoromethyl,2′-deoxy-2′-trifluoromethyl, 2′-deoxy-2′-methylene,2′-deoxy-2′-fluoromethylene, 2′-deoxy-2′-difluoromethylene,2′-deoxy-2′-ethyl, 2′-deoxy-2′-ethylene and 2′-deoxy-2′-acetylene. Thesenucleotides can alternately be described as 2-SCH₃ ribonucleotide,2′-CH₃ribonucleotide, 2′-NH₂ ribonucleotide 2′-NH(C₁-C₂alkyl)ribonucleotide, 2′-N(C₁-C₂ alkyl)₂ ribonucleotide, 2′-CH₂Fribonucleotide, 2′-CHF₂ ribonucleotide, 2′-CF₃ ribonucleotide, 2′=CH₂ribonucleotide, 2′=CHF ribonucleotide, 2′=CF₂ ribonucleotide, 2′-C₂H₅ribonucleotide, 2′-CH═CH₂ ribonucleotide, 2′-C≡CH ribonucleotide. Afurther useful ribonucleotide is one having a ring located on the ribosering in a cage-like structure including3′,O,4′-C-methyleneribonucleotides. Such cage-like structures willphysically fix the ribose ring in the desired conformation.

Additionally, preferred for use as the B-form nucleotides for elicitingRNase H are arabino nucleotides having 2′-deoxy-2′-cyano,2′-deoxy-2′-fluoro, 2′-deoxy-2′-chloro, 2′-deoxy-2′-bromo,2′-deoxy-2′-azido, 2′-methoxy and the unmodified arabino nucleotide(that includes a 2′-OH projecting upwards towards the base of thenucleotide). These arabino nucleotides can alternately be described as2′-CN arabino nucleotide, 2′-F arabino nucleotide, 2′-Cl arabinonucleotide, 2′-Br arabino nucleotide, 2′-N₃ arabino nucleotide, 2′-O—CH₃arabino nucleotide and arabino nucleotide.

Such nucleotides are linked together via phosphorothioate,phosphorodithioate, boranophosphate or phosphodiester linkages.particularly preferred is the phosphorothioate linkage.

Illustrative of the B-form nucleotides for use in the invention is a2′-S-methyl (2′-SMe) nucleotide that resides in C2′ endo conformation.It can be compared to 2′-O-methyl (2′-OMe) nucleotides that resides in aC3′ endo conformation. Particularly suitable for use in comparing thesetwo nucleotides are molecular dynamic investigations using a SGI[Silicon Graphics, Mountain View, Calif.] computer and the AMBER [UCSF,San Francisco, Calif.] modeling software package for computersimulations.

Ribose conformations in C2′-modified nucleosides containing S-methylgroups were examined. To understand the influence of2 ′-O-methyl and2′-S-methyl groups on the conformation of nucleosides, we evaluated therelative energies of the 2′-O- and 2 ′-S-methylguanosine, along withnormal deoxyguanosine and riboguanosine, starting from both C2′-endo andC3′-endo conformations using ab initio quantum mechanical calculations.All the structures were fully optimized at HF/6-31G* level and singlepoint energies with electron-correlation were obtained at theMP2/6-31G*//HF/6-31G* level. As shown in Table 1, the C2′-endoconformation of deoxyguanosine is estimated to be 0.6 kcal/mol morestable than the C3′-endo conformation in the gas-phase. Theconformational preference of the C2′-endo over the C3′-endo conformationappears to be less dependent upon electron correlation as revealed bythe MP2/6-31G*//HF/6-31G* values which also predict the same differencein energy. The opposite trend is noted for riboguanosine. At theHF/6-31G* and MP2/6-31G*//HF/6-31G* levels, the C3′-endo form ofriboguanosine is shown to be about 0.65 and 1.41 kcal/mol more stablethan the C2′ endo form, respectively.

TABLE 1 Relative energies* of the C3'-endo and C2'-endo conformations ofrepresentative nucleosides. CONTINUUM HF/6-31GMP2/6-31-G MODEL AMBER dG0.60 0.56 0.88 0.65 rG −0.65 −1.41 −0.28 −2.09 2'-O-MeG −0.89 −1.79−0.36 −0.86 2'-S-MeG 2.55 1.41 3.16 2.43 *energies are in kcal/molrelative to the C2'-endo confonnation

Table 1 also includes the relative energies of 2′-O-methylguanosine and2′-S-methylguanosine in C2′-endo and C3′-endo conformation. This dataindicates the electronic nature of C2′-substitution has a significantimpact on the relative stability of these conformations. Substitution ofthe 2′-O-methyl group increases the preference for the C3′-endoconformation (when compared to riboguanosine) by about 0.4 kcal/mol atboth the HF/6-31G* and MP2/6-31G*//HF16-31G* levels. In contrast, the2′-S-methyl group reverses the trend. The C2′-endo conformation isfavored by about 2.6 kcal/mol at the HF/6-31G* level, while the samedifference is reduced to 1.41 kcal/mol at the MP2/6-31G*//HF/6-31G*level. For comparison, and also to evaluate the accuracy of themolecular mechanical force-field parameters used for the 2′-O-methyl and2′-S-methyl substituted nucleosides, we have calculated the gas phaseenergies of the nucleosides. The results reported in Table 1 indicatethat the calculated relative energies of these nucleosides comparequalitatively well with the ab initio calculations.

Additional calculations were also performed to gauge the effect ofsolvation on the relative stability of nucleoside conformations. Theestimated solvation effect using HF/6-31G* geometries confirms that therelative energetic preference of the four nucleosides in the gas-phaseis maintained in the aqueous phase as well (Table 1). Solvation effectswere also examined using molecular dynamics simulations of thenucleosides in explicit water. From these trajectories, one can observethe predominance of C2′-endo conformation for deoxyriboguanosine and2′-S-methylriboguanosine while riboguanosine and2′-O-methylriboguanosine prefer the C3′-endo conformation. These resultsare in much accord with the available NMR results on2′-S-methylribonucleosides. NMR studies of sugar puckering equilibriumusing vicinal spin-coupling constants have indicated that theconformation of the sugar ring in 2′-S-methylpyrimidine nucleosides showan average of >75% S-character, whereas the corresponding purine analogsexhibit an average of >90% S-pucker [Fraser, A., Wheeler, P., Cook, P.D. and Sanghvi, Y. S., J. Heterocycl. Chem., 1993, 30, 1277-1287]. Itwas observed that the 2′-S-methyl substitution in deoxynucleosideconfers more conformational rigidity to the sugar conformation whencompared with deoxyribonucleosides.

Structural features of DNA:RNA, OMe_DNA:RNA and SMe_DNA:RNA hybrids werealso observed. The average RMS deviation of the DNA:RNA structure fromthe starting hybrid coordinates indicate the structure is stabilizedover the length of the simulation with an approximate average RMSdeviation of 1.0 Å. This deviation is due, in part, to inherentdifferences in averaged structures (i.e. the starting conformation) andstructures at thermal equilibrium. The changes in sugar puckerconformation for three of the central base pairs of this hybrid are ingood agreement with the observations made in previous NMR studies. Thesugars in the RNA strand maintain very stable geometries in the C3′-endoconformation with ring pucker values near 0°. In contrast, the sugars ofthe DNA strand show significant variability.

The average RMS deviation of the OMe_DNA:RNA is approximately 1.2 Å fromthe starting A-form conformation; while the SMe_DNA:RNA shows a slightlyhigher deviation (approximately 1.8 Å) from the starting hybridconformation. The SMe_DNA strand also shows a greater variance in RMSdeviation, suggesting the S-methyl group may induce some structuralfluctuations. The sugar puckers of the RNA complements maintain C3′-endopuckering throughout the simulation. As expected from the nucleosidecalculations, however, significant differences are noted in thepuckering of the OMe_DNA and SMe_DNA strands, with the former adoptingC3′-endo, and the latter, C1′-exo/C2′-endo conformations.

An analysis of the helicoidal parameters for all three hybrid structureshas also been performed to further characterize the duplex conformation.Three of the more important axis-basepair parameters that distinguishthe different forms of the duplexes, X-displacement, propeller twist,and inclination, are reported in Table 2. Usually, an X-displacementnear zero represents a B-form duplex; while a negative displacement,which is a direct measure of deviation of the helix from the helicalaxis, makes the structure appear more A-like in conformation. In A-formduplexes, these values typically vary from −4 Å to −5 Å. In comparingthese values for all three hybrids, the SMe_DNA:RNA hybrid shows themost deviation from the A-form value, the OMe_DNA:RNA shows the least,and the DNA:RNA is intermediate. A similar trend is also evident whencomparing the inclination and propeller twist values with ideal A-formparameters. These results are further supported by an analysis of thebackbone and glycosidic torsion angles of the hybrid structures:Glycosidic angles (X) of A-form geometries, for example, are typicallynear −159° while B form values are near −102°. These angles are found tobe −162°, −133°, and −108° for the OMe_DNA, DNA, and SMe_DNA strands,respectively. All RNA complements adopt an X angle close to −160°. Inaddition, “crankshaft” transitions were also noted in the backbonetorsions of the central UpU steps of the RNA strand in the SMe_DNA:RNAand DNA;RNA hybrids. Such transitions suggest some local conformationalchanges may occur to relieve a less favorable global conformation. Takenoverall, the results indicate the amount of A-character decreases asOMe_DNA:RNA>DNA:RNA>SMe_DNA:RNA, with the latter two adopting moreintermediate conformations when compared to A- and B-form geometries.

TABLE 2 Average helical parameters derived from the last 500 ps ofsimulation time. (canonical A- and B- form values are given forcomparison) OMe_(—) SMe_(—) Helicoidal B-DNA B-DNA A-DNA DNA: DNA: DNA:Parameter (x-ray) (fibre) (fibre) RNA RNA RNA X-disp 1.2 0.0 −5.3 −4.5−5.4 −3.5 Inclination −2.3 1.5 20.7 11.6 15.1 0.7 Propeller −16.4 −13.3−7.5 −12.7 −15.8 −10.3

Stability of C2′-modified DNA:RNA hybrids was determined. Although theoverall stability of the DNA:RNA hybrids depends on several factorsincluding sequence-dependencies and the purine content in the DNA or RNAstrands DNA:RNA hybrids are usually less stable than RNA:RNA duplexesand, in some cases, even less stable than DNA:DNA duplexes. Availableexperimental data attributes the relatively lowered stability of DNA:RNAhybrids largely to its intermediate conformational nature betweenDNA:DNA (B-family) and RNA:RNA (A-family) duplexes. The overallthermodynamic stability of nucleic acid duplexes may originate fromseveral factors including the conformation of backbone, base-pairing andstacking interactions. While it is difficult to ascertain the individualthermodynamic contributions to the overall stabilization of the duplex,it is reasonable to argue that the major factors that promote increasedstability of hybrid duplexes are better stacking interactions(electrostatic π-π_interactions) and more favorable groove dimensionsfor hydration. The C2′-S-methyl substitution has been shown todestabilize the hybrid duplex. The notable differences in the risevalues among the three hybrids may offer some explanation. While the2′-S-methyl group has a strong influence on decreasing the base-stackingthrough high rise values (˜3.2 Å), the 2′-O-methyl group makes theoverall structure more compact with a rise value that is equal to thatof A-form duplexes (˜2.6 Å). Despite its overall A-like structuralfeatures, the SMe_DNA:RNA hybrid structure possesses an average risevalue of 3.2 Å which is quite close to that of B-family duplexes. Infact, some local base-steps (CG steps) may be observed to have unusuallyhigh rise values (as high as 4.5 Å). Thus, the greater destabilizationof 2′-S-methyl substituted DNA:RNA hybrids may be partly attributed topoor stacking interactions.

It has been postulated that RNase H binds to the minor groove of RNA:DNAhybrid complexes, requiring an intermediate minor groove width betweenideal A- and B-form geometries to optimize interactions between thesugar phosphate backbone atoms and RNase H. A close inspection of theaveraged structures for the hybrid duplexes using computer simulationsreveals significant variation in the minor groove width dimensions asshown in Table 3. Whereas the O-methyl substitution leads to a slightexpansion of the minor groove width when compared to the standardDNA:RNA complex, the S-methyl substitution leads to a generalcontraction (approximately 0.9 Å). These changes are most likely due tothe preferred sugar puckering noted for the antisense strands whichinduce either A- or B-like single strand conformations. In addition tominor groove variations, the results also point to potential differencesin the steric makeup of the minor groove. The O-methyl group points intothe minor groove while the S-methyl is directed away towards the majorgroove. Essentially, the S-methyl group has flipped through the basesinto the major groove as a consequence of C2′-endo puckering.

TABLE 3 Minor groove widths averaged over the last 500 ps of simulationtime OMe_(—) SMe_(—) Phosphate DNA: DNA: DNA: DNA:RNA RNA:RNA DistanceRNA RNA RNA (B-form) (A-form) P5-P20 15.27 16.82 13.73 14.19 17.32P6-P19 15.52 16.79 15.73 12.66 17.12 P7-P18 15.19 16.40 14.08 11.1016.60 P8-P17 15.07 16.12 14.00 10.98 16.14 P9-P16 15.29 16.25 14.9811.65 16.93 P10-P15 15.37 16.57 13.92 14.05 17.69

In addition to the modifications described above, the nucleotides of theoligonucleotides of the invention can have a variety of othermodification so long as these other modifications do not significantlydetract from the properties described above. Thus, for nucleotides thatare incorporated into oligonucleotides of the invention, thesenucleotides can have sugar portions that correspond tonaturally-occurring sugars or modified sugars. Representative modifiedsugars include carbocyclic or acyclic sugars, sugars having substituentgroups at their 2′ position, sugars having substituent groups at their3′ position, and sugars having substituents in place of one or morehydrogen atoms of the sugar. Other altered base moieties and alteredsugar moieties are disclosed in U.S. Pat. No. 3,687,808 and PCTapplication PCT/US89/02323.

Altered base moieties or altered sugar moieties also include othermodifications consistent with the spirit of this invention. Sucholigonucleotides are best described as being structurallydistinguishable from, yet functionally interchangeable with, naturallyoccurring or synthetic wild type oligonucleotides. All sucholigonucleotides are comprehended by this invention so long as theyfunction effectively to mimic the structure of a desired RNA or DNAstrand. A class of representative base modifications include tricycliccytosine analog, termed “G clamp” (Lin, et al., J. Am. Chem. Soc. 1998,120, 8531). This analog makes four hydrogen bonds to a complementaryguanine (G) within a helix by simultaneously recognizing theWatson-Crick and Hoogsteen faces of the targeted G. This G clampmodification when incorporated into phosphorothioate oligonucleotides,dramatically enhances antisense potencies in cell culture. Theoligonucleotides of the invention also can includephenoxazine-substituted bases of the type disclosed by Flanagan, et al.,Nat. Biotechnol. 1999, 17(1), 48-52.

Additional modifications may also be made at other positions on theoligonucleotide, particularly the 3′position of the sugar on the3′terminal nucleotide and the 5′ position of 5′ terminal nucleotide. Forexample, one additional modification of the oligonucleotides of theinvention involves chemically linking to the oligonucleotide one or moremoieties or conjugates which enhance the activity, cellular distributionor cellular uptake of the oligonucleotide. Such moieties include but arenot limited to lipid moieties such as a cholesterol moiety (Letsinger etal., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553), cholic acid (Manoharanet al., Bioorg. Med. Chem. Lett., 1994, 4, 1053), a thioether, e.g.,hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y Acad. Sci., 1992, 660,306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765), athiocholesterol (Oberhauser et al., Nucl Acids Res., 1992, 20, 533), analiphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaraset al., EMBO J., 1991, 10, 111; Kabanov et al., FEBS Lett.,1990,259,327; Svinarchuk et al., Biochimie, 1993, 75, 49), aphospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990,18, 3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14, 969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277, 923).

As used herein, the term “alkyl” includes but is not limited to straightchain, branch chain, and cyclic unsaturated hydrocarbon groups includingbut not limited to methyl, ethyl, and isopropyl groups. Alkyl groups ofthe present invention may be substituted. Representative alkylsubstituents are disclosed in U.S. Pat. No. 5,212,295, at column 12,lines 41-50, hereby incorporated by reference in its entirety.

Alkenyl groups according to the invention are to straight chain, branchchain, and cyclic hydrocarbon groups containing at least onecarbon-carbon double bond, and alkynyl groups are to straight chain,branch chain, and cyclic hydrocarbon groups containing at least onecarbon-carbon triply bond. Alkenyl and alkynyl groups of the presentinvention can be substituted.

Aryl groups are substituted and unsubstituted aromatic cyclic moietiesincluding but not limited to phenyl, naphthyl, anthracyl, phenanthryl,pyrenyl, and xylyl groups. Alkaryl groups are those in which an arylmoiety links an alkyl moiety to a core structure, and aralkyl groups arethose in which an alkyl moiety links an aryl moiety to a core structure.

In general, the term “hetero” denotes an atom other than carbon,preferably but not exclusively N, O, or S. Accordingly, the term“heterocyclic ring” denotes a carbon-based ring system having one ormore heteroatoms (i.e., non-carbon atoms). Preferred heterocyclic ringsinclude, for example but not limited to imidazole, pyrrolidine,1,3-dioxane, piperazine, morpholine rings. As used herein, the term“heterocyclic ring” also denotes a ring system having one or more doublebonds, and one or more heteroatoms. Preferred heterocyclic ringsinclude, for example but not limited to the pyrrolidino ring.

Oligonucleotides according to the present invention that arehybridizable to a target nucleic acid preferably comprise from about 5to about 50 nucleosides. It is more preferred that such compoundscomprise from about 8 to about 30 nucleosides, with 15 to 25 nucleosidesbeing particularly preferred. As used herein, a target nucleic acid isany nucleic acid that can hybridize with a complementary nucleicacid-like compound. Further in the context of this invention,“hybridization” shall mean hydrogen bonding, which may be Watson-Crick,Hoogsteen or reversed Hoogsteen hydrogen bonding between complementarynucleobases. “Complementary” as used herein, refers to the capacity forprecise pairing between two nucleobases. For example, adenine andthymine are complementary nucleobases which pair through the formationof hydrogen bonds. “Complementary” and “specifically hybridizable,” asused herein, refer to precise pairing or sequence complementaritybetween a first and a second nucleic acid-like oligomers containingnucleoside subunits. For example, if a nucleobase at a certain positionof the first nucleic acid is capable of hydrogen bonding with anucleobase at the same position of the second nucleic acid, then thefirst nucleic acid and the second nucleic acid are considered to becomplementary to each other at that position. The first and secondnucleic acids are complementary to each other when a sufficient numberof corresponding positions in each molecule are occupied by nucleobaseswhich can hydrogen bond with each other. Thus, “specificallyhybridizable” and “complementary” are terms which are used to indicate asufficient degree of complementarity such that stable and specificbinding occurs between a compound of the invention and a target RNAmolecule. It is understood that an oligomeric compound of the inventionneed not be 100% complementary to its target RNA sequence to bespecifically hybridizable. An oligomeric compound is specificallyhybridizable when binding of the oligomeric compound to the target RNAmolecule interferes with the normal function of the target RNA to causea loss of utility, and there is a sufficient degree of complementarityto avoid non-specific binding of the oligomeric compound to non-targetsequences under conditions in which specific binding is desired, i.e.under physiological conditions in the case of in vivo assays ortherapeutic treatment, or in the case of in vitro assays, underconditions in which the assays are performed.

The oligonucleotides of the invention can be used in diagnostics,therapeutics and as research reagents and kits. They can be used inpharmaceutical compositions by including a suitable pharmaceuticallyacceptable diluent or carrier. They further can be used for treatingorganisms having a disease characterized by the undesired production ofa protein. The organism should be contacted with an oligonucleotidehaving a sequence that is capable of specifically hybridizing with astrand of nucleic acid coding for the undesirable protein. Treatments ofthis type can be practiced on a variety of organisms ranging fromunicellular prokaryotic and eukaryotic organisms to multicellulareukaryotic organisms. Any organism that utilizes DNA-RNA transcriptionor RNA-protein translation as a fundamental part of its hereditary,metabolic or cellular control is susceptible to therapeutic and/orprophylactic treatment in accordance with the invention. Seeminglydiverse organisms such as bacteria, yeast, protozoa, algae, all plantsand all higher animal forms, including warm-blooded animals, can betreated. Further, each cell of multicellular eukaryotes can be treated,as they include both DNA-RNA transcription and RNA-protein translationas integral parts of their cellular activity. Furthermore, many of theorganelles (e.g. mitochondria and chloroplasts) of eukaryotic cells alsoinclude transcription and translation mechanisms. Thus, single cells,cellular populations or organelles can also be included within thedefinition of organisms that can be treated with therapeutic ordiagnostic oligonucleotides.

Some representative therapeutic indications and other uses for thecompounds of the invention are as follows:

One therapeutic indication of particular interest is psoriasis.Psoriasis is a common chronic and recurrent disease characterized bydry, well-circumscribed, silvery, scaling papules and plaques of varioussizes. The disease varies in severity from a few lesions to widespreaddermatosis with disabling arthritis or exfoliation. The ultimate causeof psoriasis is not known, but the thick scaling that occurs is probablydue to increased epidermal cell proliferation (The Merck Manual ofDiagnosis and Therapy, 15th Ed., pp. 2283-2285, Berkow et al., eds.,Rahway, N.J., 1987). Inhibitors of Protein Kinase C (PKC) have beenshown to have both antiproliferative and anti-inflammatory effects invitro. Some antipsoriasis drugs, such as cyclosporin A and anthralin,have been shown to inhibit PKC, and inhibition of PKC has been suggestedas a therapeutic approach to the treatment of psoriasis (Hegemann, L.and G. Mahrle, Pharmacology of the Skin, H. Mukhtar, ed., pp. 357-368,CRC Press, Boca Raton, Fla., 1992). Antisense compounds targeted toProtein Kinase C (PKC) proteins are described in U.S. Pat. No. 5,620,963to Cook et al. and U.S. Pat. No. 5,681,747 to Boggs et al.

Another type of therapeutic indication of interest is inflammatorydisorders of the skin. These occur in a variety of forms including, forexample, lichen planus, toxic epidermal necrolyis (TEN), ertythemamultiforme and the like (The Merck Manual of Diagnosis and Therapy, 15thEd., pp. 2286-2292, Berkow et al., eds., Rahway, N.J., 1987). Expressionof ICAM-1 has been associated with a variety of inflammatory skindisorders such as allergic contact dermatitis, fixed drug eruption,lichen planus and psoriasis (Ho et al., J. Am. Acad. Dermatol., 1990,22, 64; Griffiths et al., Am. J. Pathology, 1989, 135, 1045; Lisby etal., Br. J. Dermatol., 1989, 120, 479; Shiohara et al., Arch. Dermatol.,1989, 125, 1371; Regezi et al., Oral Surg. Oral Med. Oral Pathol., 1996,81, 682). Moreover, intraperitoneal administration of a monoclonalantibody to ICAM-1 decreases ovalbumin-induced eosinophil infiltrationinto skin in mice (Hakugawa et al., J. Dermatol., 1997, 24,73).Antisense compounds targeted to ICAM-1 are described in U.S. Pat. Nos.5,514,788 and 5,591,623, and co-pending U.S. patent application Ser.Nos. 09/009,490 and 09/062,416, Jan. 20, 1998 and Apr. 17, 1998,respectively, all to Bennett et al.

Other antisense targets for skin inflammatory disorders are VCAM-1 andPECAM-1. Intraperitoneal administration of a monoclonal antibody toVCAM-1 decreases ovalbumin-induced eosinophil infiltration into the skinof mice (Hakugawa et al., J. Dermatol., 1997, 24, 73). Antisensecompounds targeted to VCAM-1 are described in U.S. Pat. Nos. 5,514,788and 5,591,623. PECAM-1 proteins are glycoproteins which are expressed onthe surfaces of a variety of cell types (for reviews, see Newman, J.Clin. Invest., 1997, 99, 3 and DeLisser et al., Immunol. Today, 1994,15, 490). In addition to directly participating in cell-cellinteractions, PECAM-1 apparently also regulates the activity and/orexpression of other molecules involved in cellular interactions (Litwinet al., J. Cell Biol., 1997, 139, 219) and is thus a key mediator ofseveral cell:cell interactions. Antisense compounds targeted to PECAM-1are described in co-pending U.S. patent application Ser. No. 09/044,506,filed Mar. 19, 1998, by Bennett et al.

Another type of therapeutic indication of interest for oligonucleotidesencompasses a variety of cancers of the skin. Representative skincancers include benign tumors (warts, moles and the like) and malignanttumors such as, for example, basal cell carcinoma, squamous cellcarcinoma, malignant melanoma, Paget's disease, Kaposi's sarcoma and thelike (The Merci Manual of Diagnosis and Therapy, 15th Ed., pp.2301-2310, Berkow et al., eds., Rahway, N.J., 1987). A number ofmolecular targets involved in tumorigenesis, maintenance of thehyperproliferative state and metastasis are targeted to prevent orinhibit skin cancers, or to prevent their spread to other tissues.

The ras oncogenes are guanine-binding proteins that have been implicatedin cancer by, e.g., the fact that activated ras oncogenes have beenfound in about 30% of human tumors generally; this figure approached100% in carcinomas of the exocrine pancreas (for a review, see Downward,Trends in Biol. Sci., 1990, 15, 469). Antisense compounds targeted toH-ras and K-ras are described in U.S. Pat. No. 5,582,972 to Lima et al.,U.S. Pat. No. 5,582,986 to Monia et al. and U.S. Pat. No. 5,661,134 toCook et al., and in published PCT application WO 94/08003.

Protein Kinase C (PKC) proteins have also been implicated intumorigenesis. Antisense compounds targeted to Protein Kinase C (PKC)proteins are described in U.S. Pat. No. 5,620,963 to Cook et al. andU.S. Pat. No. 5,681,747 to Boggs et al. Also of interest are AP-1subunits and JNK proteins, particularly in regard to their roles intumorigenesis and metastasis. The process of metastasis involves asequence of events wherein (1) a cancer cell detaches from itsextracellular matrices, (2) the detached cancer cell migrates to anotherportion of an animal's body, often via the circulatory system, and (3)attaches to a distal and inappropriate extracellular matrix, therebycreated a focus from which a secondary tumor can arise. Normal cells donot possess the ability to invade or metastasize and/or undergoapoptosis (programmed cell death) if such events occur (Ruoslahti, Sci.Amer., 1996, 275, 72). However, many human tumors have elevated levelsof activity of one or more matrix metalloproteinases (MMPs)(Stetler-Stevenson et al., Annu. Rev. Cell Biol., 1993, 9, 541; Bernhardet al., Proc. Natl. Acad. Sci. (U.S.A.), 1994,91, 4293. The MMPs are afamily of enzymes which have the ability to degrade components of theextracellular matrix (Birkedal-Hansen, Current Op. Biol., 1995, 7, 728).In particular, one member of this family, matrix metalloproteinase-9(MMP-9), is often found to be expressed only in tumors and otherdiseased tissues (Himelstein et al., Invasion & Metastasis, 1994, 14,246).

Several studies have shown that regulation of the MMP-9 gene may becontrolled by the AP-1 transcription factor (Kerr et al., Science, 1988,242, 1242; Kerr et al., Cell, 1990, 61, 267; Gum et al., J. Biol. Chem.,1996, 271, 10672; Hua et al., Cancer Res., 1996, 56, 5279). Inhibitionof AP-1 function has been shown to attenuate MMP-9 expression (U.S.patent application Ser. No. 08/837,201). AP-1 is a heterodimeric proteinhaving two subunits, the gene products of fos and jun. Antisensecompounds targeted to c-fos and c-jun are described in co-pending U.S.patent application Ser. No. 08/837,201, filed Mar. 14, 1997, by Dean etal.

Furthermore, AP-1 is itself activated in certain circumstances byphosphorylation of the Jun subunit at an amino-terminal position by JunN-terminal kinases (JNKs). Thus, inhibition of one or more JNKs isexpected to result in decreased AP-1 activity and, consequentially,reduced MMP expression. Antisense compounds targeted to JNKs aredescribed in co-pending U.S. patent application Ser. No. 08/910,629,filed Aug. 13, 1997, by Dean et al.

Infectious diseases of the skin are caused by viral, bacterial or fungalagents. In the case of Lyme disease, the tick borne causative agentthereof, the spirochete Borrelia burgdorferi, up-regulates theexpression of ICAM-1, VCAM-1 and ELAM-1 on endothelial cells in vitro(Boggemeyer et al., Cell Adhes. Comm., 1994, 2, 145). Furthermore, ithas been proposed that the mediation of the disease by theanti-inflammatory agent prednisolone is due in part to mediation of thisup-regulation of adhesion molecules (Hurtenbach et al., Int. J.Immunopharmac., 1996, 18, 281). Thus, potential targets for therapeuticmediation (or prevention) of Lyme disease include ICAM-1, VCAM-1 andELAM-1 (supra).

Other infectious disease of the skin which are tractable to treatmentusing the compositions and methods of the invention include disordersresulting from infection by bacterial, viral or fungal agents (The MerckManual of Diagnosis and Therapy, 15th Ed., pp. 2263-2277, Berkow et al.,eds., Rahway, N.J., 1987). With regards to infections of the skin causedby fungal agents, U.S. Pat. No. 5,691,461 provides antisense compoundsfor inhibiting the growth of Candida albicans.

With regards to infections of the skin caused by viral agents, U.S. Pat.Nos. 5,166,195, 5,523,389 and 5,591,600 provide oligonucleotideinhibitors of Human Immunodeficiency Virus (HIV). U.S. Pat. No.5,004,810 provides oligomers capable of hybridizing to herpes simplexvirus Vmw65 mRNA and inhibiting its replication. U.S. Pat. Nos.5,194,428 and 5,580,767 provide antisense compounds having antiviralactivity against influenza virus. U.S. Pat. No. 4,806,463 providesantisense compounds and methods using them to inhibit HTLV-IIIreplication. U.S. Pat. Nos. 4,689,320, 5,442,049, 5,591,720 and5,607,923 are directed to antisense compounds as antiviral agentsspecific to cytomegalovirus (CMV). U.S. Pat. No. 5,242,906 providesantisense compounds useful in the treatment of latent Epstein-Barr virus(EBV) infections. U.S. Pat. Nos. 5,248,670, 5,514,577 and 5,658,891provide antisense compounds useful in the treatment of herpes virusinfections. U.S. Pat. Nos. 5,457,189 and 5,681,944 provide antisensecompounds useful in the treatment of papilloma virus infections. Theantisense compounds disclosed in these patents, which are hereinincorporated by reference, may be used with the compositions of theinvention to effect prophylactic, palliative or therapeutic relief fromdiseases caused or exacerbated by the indicated pathogenic agents.

Antisense oligonucleotides employed in the compositions of the presentinvention may also be used to determine the nature, function andpotential relationship of various genetic components of the body todisease or body states in animals. Heretofore, the function of a genehas been chiefly examined by the construction of loss-of-functionmutations in the gene (i. e., “knock-out” mutations) in an animal (e.g.,a transgenic mouse). Such tasks are difficult, time-consuming and cannotbe accomplished for genes essential to animal development since the“knock-out” mutation would produce a lethal phenotype. Moreover, theloss-of-function phenotype cannot be transiently introduced during aparticular part of the animal's life cycle or disease state; the“knock-out” mutation is always present. “Antisense knockouts,” that is,the selective modulation of expression of a gene by antisenseoligonucleotides, rather than by direct genetic manipulation, overcomesthese limitations (see, for example, Albert et al., Trends inPharmacological Sciences, 1994, 15, 250). In addition, some genesproduce a variety of mRNA transcripts as a result of processes such asalternative splicing; a “knock-out” mutation typically removes all formsof mRNA transcripts produced from such genes and thus cannot be used toexamine the biological role of a particular mRNA transcript. Antisenseoligonucleotides have been systemically administered to rats in order tostudy the role of the N-methyl-D-aspartate receptor in neuronal death,to mice in order to investigate the biological role of protein kinaseC-a, and to rats in order to examine the role of the neuropeptide Y1receptor in anxiety (Wahlestedt et al., Nature, 1993, 363:260; Dean etal., Proc. Natl. Acad. Sci. U.S.A., 1994, 91:11762; and Wahlestedt etal., Science, 1993, 259:528, respectively). In instances where complexfamilies of related proteins are being investigated, “antisenseknockouts” (i.e., inhibition of a gene by systemic administration ofantisense oligonucleotides) may represent the most accurate means forexamining a specific member of the family (see, generally, Albert etal., Trends Pharmacol Sci., 1994, 15:250). By providing compositions andmethods for the simple non-parenteral delivery of oligonucleotides andother nucleic acids, the present invention overcomes these and othershortcomings.

The administration of therapeutic or pharmaceutical compositionscomprising the oligonucleotides of the invention is believed to bewithin the skill of those in the art. In general, a patient in need oftherapy or prophylaxis is administered a composition comprising acompound of the invention, commonly in a pharmaceutically acceptablecarrier, in doses ranging from 0.01 ug to 100 g per kg of body weightdepending on the age of the patient and the severity of the disorder ordisease state being treated. Dosing is dependent on severity andresponsiveness of the disease state to be treated, with the course oftreatment lasting from several days to several months, or until a cureis effected or a diminution or prevention of the disease state isachieved. Optimal dosing schedules can be calculated from measurementsof drug accumulation in the body of the patient. Persons of ordinaryskill can easily determine optimum dosages, dosing methodologies andrepetition rates. Optimum dosages may vary depending on the relativepotency of individual antisense compounds, and can generally beestimated based on EC₅₀s found to be effective in in vitro and in vivoanimal models.

In the context of the invention, the term “treatment regimen” is meantto encompass therapeutic, palliative and prophylactic modalities ofadministration of one or more compositions of the invention. Aparticular treatment regimen may last for a period of time which willvary depending upon the nature of the particular disease or disorder,its severity and the overall condition of the patient, and may extendfrom once daily to once every 20 years. Following treatment, the patientis monitored for changes in his/her condition and for alleviation of thesymptoms of the disorder or disease state. The dosage of the compositionmay either be increased in the event the patient does not respondsignificantly to current dosage levels, or the dose may be decreased ifan alleviation of the symptoms of the disorder or disease state isobserved, or if the disorder or disease state has been ablated.

An optimal dosing schedule is used to deliver a therapeuticallyeffective amount of the oligonucleotide of the invention. The term“therapeutically effective amount,” for the purposes of the invention,refers to the amount of oligonucleotide-containing pharmaceuticalcomposition which is effective to achieve an intended purpose withoutundesirable side effects (such as toxicity, irritation or allergicresponse). Although individual needs may vary, determination of optimalranges for effective amounts of pharmaceutical compositions is withinthe skill of the art. Human doses can be extrapolated from animalstudies (Katocs et al., Chapter 27 In: Remington's PharmaceuticalSciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa.,1990). Generally, the dosage required to provide an effective amount ofa pharmaceutical composition, which can be adjusted by one skilled inthe art, will vary depending on the age, health, physical condition,weight, type and extent of the disease or disorder of the recipient,frequency of treatment, the nature of concurrent therapy (if any) andthe nature and scope of the desired effect(s) (Nies et al., Chapter 3In: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9thEd., Hardman et al., eds., McGraw-Hill, New York, N.Y., 1996).

Following successful treatment, it may be desirable to have the patientundergo maintenance therapy to prevent the recurrence of the diseasestate, wherein the bioactive agent is administered in maintenance doses,ranging from 0.01 ug to 100 g per kg of body weight, once or more daily,to once every 20 years. For example, in the case of in individual knownor suspected of being prone to an autoimmune or inflammatory condition,prophylactic effects may be achieved by administration of preventativedoses, ranging from 0.01 ug to 100 g per kg of body weight, once or moredaily, to once every 20 years. In like fashion, an individual may bemade less susceptible to an inflammatory condition that is expected tooccur as a result of some medical treatment, e.g., graft versus hostdisease resulting from the transplantation of cells, tissue or an organinto the individual.

Prophylactic modalities for high risk individuals are also encompassedby the invention. As used herein, the term “high risk individual” ismeant to refer to an individual for whom it has been determined, via,e.g., individual or family history or genetic testing, that there is asignificantly higher than normal probability of being susceptible to theonset or recurrence of a disease or disorder. For example, a subjectanimal could have a personal and/or family medical history that includesfrequent occurrences of a particular disease or disorder. As anotherexample, a subject animal could have had such a susceptibilitydetermined by genetic screening according to techniques known in the art(see, e.g., U.S. Congress, Office of Technology Assessment, Chapter 5In: Genetic Monitoring and Screening in the Workplace, OTA-BA-455, U.S.Government Printing Office, Washington, D.C., 1990, pages 75-99). Aspart of a treatment regimen for a high risk individual, the individualcan be prophylactically treated to prevent the onset or recurrence ofthe disease or disorder. The term “prophylactically effective amount” ismeant to refer to an amount of a pharmaceutical composition whichproduces an effect observed as the prevention of the onset or recurrenceof a disease or disorder. Prophylactically effective amounts of apharmaceutical composition are typically determined by the effect theyhave compared to the effect observed when a second pharmaceuticalcomposition lacking the active agent is administered to a similarlysituated individual.

For therapeutic use the oligonucleotide analog is administered to ananimal suffering from a disease modulated by some protein. It ispreferred to administer to patients suspected of suffering from such adisease an amount of oligonucleotide analog that is effective to reducethe symptomology of that disease. One skilled in the art can determineoptimum dosages and treatment schedules for such treatment regimens.

For use in diseases modulated by protein that portion of DNA or RNAwhich codes for the protein whose formation or activity is to bemodulated is targeted. The targeting portion of the composition to beemployed is, thus, selected to be complementary to the preselectedportion of DNA or RNA, that is to be an antisense oligonucleotide forthat portion.

It is generally preferred to administer the therapeutic agents inaccordance with this invention internally such as orally, intravenously,or intramuscularly. Other forms of administration, such astransdermally, topically, or intralesionally may also be useful.Inclusion in suppositories may also be useful. Use of pharmacologicallyacceptable carriers is also preferred for some embodiments.

This invention is also directed to methods for the selective binding ofRNA for research and diagnostic purposes wherein it is useful to effectstrand cleavage utilizing enzymatic RNase H cleavage while concurrentlyeffecting modulation of binding affinity and or nuclease resistance.Such selective is accomplished by interacting such RNA or DNA withcompositions of the invention which are resistant to degradativenucleases and which hybridize more strongly and with greater fidelitythan known oligonucleotides or oligonucleotide analogs.

Oligonucleotides according to the invention can be assembled in solutionor through solid-phase reactions, for example, on a suitable DNAsynthesizer utilizing nucleosides, phosphoramidites and derivatizedcontrolled pore glass (CPG) according to the invention and/or standardnucleotide precursors. In addition to nucleosides that include a novelmodification of the inventions other nucleoside within anoligonucleotide may be further modified with other modifications at the2′ position. Precursor nucleoside and nucleotide precursors used to formsuch additional modification may carry substituents either at the 2′ or3′ positions. Such precursors may be synthesized according to thepresent invention by reacting appropriately protected nucleosidesbearing at least one free 2′ or 3′ hydroxyl group with an appropriatealkylating agent such as, but not limited to, alkoxyalkyl halides,alkoxylalkylsulfonates, hydroxyalkyl halides, hydroxyalkyl sulfonates,aminoalkyl halides, aminoalkyl sulfonates, phthalimidoalkyl halides,phthalimidoalkyl sulfonates, alkylaminoalkyl halides, alkylaminoalkylsulfonates, dialkylaminoalkyl halides, dialkylaminoalkylsulfonates,dialkylaminooxyalkyl halides, dialkylaminooxyalkyl sulfonates andsuitably protected versions of the same. Preferred halides used foralkylating reactions include chloride, bromide, fluoride and iodide.Preferred sulfonate leaving groups used for alkylating reactionsinclude, but are not limited to, benzenesulfonate, methylsulfonate,tosylate, p-bromobenzenesulfonate, triflate, trifluoroethylsulfonate,and (2,4-dinitroanilino)benzenesulfonate.

Suitably protected nucleosides can be assembled into oligonucleotidesaccording to known techniques. See, for example, Beaucage et al.,Tetrahedron, 1992, 48, 2223.

The ability of oligonucleotides to bind to their complementary targetstrands is compared by determining the melting temperature (T_(m)) ofthe hybridization complex of the oligonucleotide and its complementarystrand. The melting temperature (T_(m)), a characteristic physicalproperty of double helices, denotes the temperature (in degreescentigrade) at which 50% helical (hybridized) versus coil (unhybridized)forms are present. T_(m) is measured by using the UV spectrum todetermine the formation and breakdown (melting) of the hybridizationcomplex. Base stacking, which occurs during hybridization, isaccompanied by a reduction in UV absorption (hypochromicity).Consequently, a reduction in UV absorption indicates a higher T_(m). Thehigher the T_(m), the greater the strength of the bonds between thestrands. The structure-stability relationships of a large number ofnucleic acid modifications have been reviewed (Freier and Altmann, Nucl.Acids Research, 1997, 25, 4429-443).

The relative binding ability of the oligonucleotides of the presentinvention was determined using protocols as described in the literature(Freier and Altmann, Nucl. Acids Research, 1997, 25, 4429-443).Typically absorbance versus temperature curves were determined usingsamples containing 4 uM oligonucleotide in 100 mM Na+, 10 mM phosphate,0.1 mM EDTA, and 4 uM complementary, length matched RNA.

The in vivo stability of oligonucleotides is an important factor toconsider in the development of oligonucleotide therapeutics. Resistanceof oligonucleotides to degradation by nucleases, phosphodiesterases andother enzymes is therefore determined. Typical in vivo assessment ofstability of the oligonucleotides of the present invention is performedby administering a single dose of 5 mg/kg of oligonucleotide inphosphate buffered saline to BALB/c mice. Blood collected at specifictime intervals post-administration is analyzed by HPLC or capillary gelelectrophoresis (CGE) to determine the amount of oligonucleotideremaining intact in circulation and the nature the of the degradationproducts.

Heterocyclic bases amenable to the present invention include bothnaturally and non-naturally occurring nucleobases and heterocycles. Arepresentative list includes adenine, guanine, cytosine, uridine, andthymine, as well as other synthetic and natural nucleobases such asxanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 5-halo uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudo uracil), 4-thiouracil, 8-halo,oxa, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adeninesand guanines, 5-trifluoromethyl and other 5-substituted uracils andcytosines, 7-methylguanine. Further heterocyclic bases include thosedisclosed in U.S. Pat. No. 3,687,808, those disclosed in the ConciseEncyclopedia Of Polymer Science And Engineering, pages 858-859,Kroschwitz, J. I., ed. John Wiley & Sons, 1990, and those disclosed byEnglisch, et al., Angewandte Chemie, International Edition 1991, 30,613.

Additional objects, advantages, and novel features of this inventionwill become apparent to those skilled in the art upon examination of thefollowing examples, which are not intended to be limiting. Alloligonucleotide sequences are listed in a standard 5′ to 3′ order fromleft to right.

EXAMPLE 1

5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyl uridine and5′-O-DMT-3′-O-(2-methoxyethyl)5-methyl uridine

2′,3′-O-dibutylstannylene 5-methyl uridine (345 g) (prepared as per:Wagner et al., J. Org. Chem., 1974, 39, 24) was alkylated with2-methoxyethyl bromide (196 g) in the presence of tetrabutylammoniumiodide (235 g) in DMF (3 L) at 70° C. to give a mixture of 2′-O- and3′-O-(2-methoxyethyl)-5-methyl uridine (150 g) in nearly 1:1 ratio ofisomers. The mixture was treated with DMT chloride (110 g, DMT-Cl) inpyridine (1 L) to give a mixture of the 5′-O-DMT-nucleosides. After thestandard work-up the isomers were separated by silica gel columnchromatography. The 2′-isomer eluted first, followed by the 3′-isomer.

EXAMPLE 2

5′-O-DMT-3′-O-(2-methoxyethyl)-5-methyl-uridine-2′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite

5′-O-DMT-3′-O-(2-methoxyethyl)-5-methyluridine (5 g, 0.008 mol) wasdissolved in CH₂Cl₂ (30 mL) and to this solution, under argon,diisopropylaminotetrazolide (0.415 g) and 2-cyanoethoxy-N,N-diisopropylphosphoramidite (3.9 mL) were added. The reaction was stirred overnight.The solvent was evaporated and the residue was applied to silica columnand eluted with ethyl acetate to give 3.75 g title compound.

EXAMPLE 3

5′-O-DMT-3′-O-methoxyethyl)-N⁴-benzoyl-5-methyl-cytidine

5′-O-DMT-3′-O-(2-methoxyethyl)-5-methyl uridine (15 g) was treated with150 mL anhydrous pyridine and 4.5 mL of acetic anhydride under argon andstirred overnight. Pyridine was evaporated and the residue waspartitioned between 200 mL of saturated NaHCO₃ solution and 200 mL ofethylacetate. The organic layer was dried (anhydrous MgSO₄) andevaporated to give 16 g of2′-acetoxy-5′-O-(DMT)-3′-O-(2-methoxyethyl)-5-methyl uridine.

To an ice-cold solution of triazole (19.9 g) in triethylamine (50 mL)and acetonitrile (150 mL), with mechanical stirring, 9 mL of POCl₃ wasadded dropwise. After the addition, the ice bath was removed and themixture stirred for 30 min. The2′-acetoxy-5′-O-(DMT)-3′-O-(2-methoxyethyl)-5-methyl uridine (16 g in 50mL CH₃CN) was added dropwise to the above solution with the receivingflask kept at ice bath temperatures. After 2 hrs, TLC indicated a fastermoving nucleoside, C-4-triazole-derivative. The reaction flask wasevaporated and the nucleoside was partitioned between ethylacetate (500mL) and NaHCO₃ (500 mL). The organic layer was washed with saturatedNaCl solution, dried (anhydrous NgSO₄) and evaporated to give 15 g ofC-4-triazole nucleoside. This compound was then dissolved in 2:1 mixtureof NH₄OH/dioxane (100 mL:200 mL) and stirred overnight. TLC indicateddisappearance of the starting material. The solution was evaporated anddissolved in methanol to crystallize out 9.6 g of5′-O-(DMT)-3′-O-(2-methoxyethyl)5-methyl cytidine.

5′-O-DMT-3′-O-(2-methoxyethyl)-5-methyl cytidine (9.6 g, 0.015 mol) wasdissolved in 50 mL of DMF and treated with 7.37 g of benzoic anhydride.After 24 hrs of stirring, DMF was evaporated and the residue was loadedon silica column and eluted with 1:1 hexane:ethylacetate to give thedesired nucleoside.

EXAMPLE 4

5′-O-DMT-3′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methyl-cytidine-2′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite

5′-O-DMT-3′-O-(2-methoxyethyl)-N-benzoyl-5-methyl-cytidine-2′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramiditewas obtained from the above nucleoside using the phosphitylationprotocol described for the corresponding 5-methyl-uridine derivative.

EXAMPLE 5

N⁶-Benzoyl-5′-O-(DMT)-3′-O-(2-methoxyethyl)adenosine

A solution of adenosine (42.74 g, 0.16 mol) in dry dimethyl formamide(800 mL) at 5° C. was treated with sodium hydride (8.24 g, 60% in oilprewashed thrice with hexanes, 0.21 mol). After stirring for 30 min,2-methoxyethyl bromide (0.16 mol) was added over 20 min. The reactionwas stirred at 5° C. for 8 h, then filtered through Celite. The filtratewas concentrated under reduced pressure followed by coevaporation withtoluene (2×10 mL). The residue was adsorbed on silica gel (100 g) andchromatographed (800 g, chloroform-methanol 9:14:1). Selected fractionswere concentrated under reduced pressure and the residue was a mixtureof 2′-O-(2-(methoxyethyl)adenosine and 3′-O-(2-methoxyethyl)adenosine inthe ratio of 4:1.

The above mixture (0.056 mol) in pyridine (100 mL) was evaporated underreduced pressure to dryness. The residue was redissolved in pyridine(560 mL) and cooled in an ice water bath. Trimethylsilyl chloride (36.4mL, 0.291 mol) was added and the reaction was stirred at 5° C. for 30min. Benzoyl chloride (33.6 mL, 0.291 mol) was added and the reactionwas allowed to warm to 25° C. for 2 h and then cooled to 5° C. Thereaction was diluted with cold water (112 mL) and after stirring for 15min, concentrated ammonium hydroxide (112 Ml) was added. After 30 min,the reaction was concentrated under reduced pressure (below 30° C.)followed by coevaporation with toluene (2×100 mL). The residue wasdissolved in ethyl acetate-methanol (400 mL, 9:1) and the undesiredsilyl by-products were removed by filtration. The filtrate wasconcentrated under reduced pressure and then chromatographed on silicagel (800 g, chloroform-methanol 9:1). Selected fractions were combined,concentrated under reduced pressure and dried at 25° C./0.2 mmHg for 2 hto give pure N⁶-Benzoyl-2′-O-(2-methoxyethyl)adenosine and pureN⁶Benzoyl-3′-O-(2-methoxyethyl)adenosine.

A solution of N⁶-Benzoyl-3′-O-(2-methoxyethyl)adenosine (11.0 g, 0.285mol) in pyridine (100 mL) was evaporated under reduced pressure to anoil. The residue was redissolved in dry pyridine (300 mL) and DMT-Cl(10.9 g, 95%, 0.31 mol) was added. The mixture was stirred at 25° C. for16 h and then poured onto a solution of sodium bicarbonate (20 g) in icewater (500 mL). The product was extracted with ethyl acetate (2×150 mL).The organic layer was washed with brine (50 mL), dried over sodiumsulfate (powdered) and evaporated under reduced pressure (below 40 C.).The residue was chromatographed on silica gel (400 g, ethylacetate-acetonitrile-triethylamine 99:1:195:5:1). Selected fractionswere combined, concentrated under reduced pressure and dried at 25°C./0.2 mmHg to give 16.8 g (73%) of the title compound as a foam. TheTLC was homogenous.

EXAMPLE 6

[N⁶-Benzoyl-5′-O(DM3′-O-(2-methoxyethyl)adenosin-2′-O-2-cyanoethyl-N,N-diisopropyl)phosphoramidite

The title compound was prepared in the same manner as the5-methyl-cytidine and 5-methyluridine analogs of Examples 2 and 4 bystarting with the title compound of Example 5. Purification using ethylacetate-hexanes-triethylamine 59:40:1 as the chromatography eluent gave67% yield of the title compound as a solid foam. The TLC was homogenous.³¹P-NMR (CDCl₃, H₃PO₄ std.) δ 147.89; 148.36 (diastereomers).

EXAMPLE 7

5′O-(DMT)-N²-isobutyryl-3′-O-(2-methoxyethyl)guanosine

A. 2.6-Diaminopurine riboside

To a 2 L stainless steel Parr bomb was added guanosine hydrate (100 g,0.35 mol, Aldrich), hexamethyl) disilazane (320 mL, 1.52 mol, 4.4 eq.),trimethyl) silyl trifiouromethanesulfonate (8.2 mL), and toluene (350mL). The bomb was sealed and partially submerged in an oil bath (170°C.; internal T 150° C., 150 psi) for 5 days. The bomb was cooled in adry ice/acetone bath and opened. The contents were transferred withmethanol (300 mL) to a flask and the solvent was evaporated underreduced pressure. Aqueous methanol (50%, 1.2 L) was added. The resultingbrown suspension was heated to reflux for 5 h. The suspension wasconcentrated under reduced pressure to one half volume in order toremove most of the methanol. Water (600 mL) was added and the solutionwas heated to reflux, treated with charcoal (5 g) and hot filteredthrough Celite. The solution was allowed to cool to 25° C. The resultingprecipitate was collected, washed with water (200 mL) and dried at 90°C./0.2 mmHg for 5 h to give a constant weight of 87.4 g (89%) of tan,crystalline solid; mp 247° C. (shrinks), 255° C. (dec, lit. (1) mp250-252° C.); TLC homogenous (Rf 0.50, isopropanol-ammoniumhydroxide-water 16:3:1 ); PMR (DMSO), δ 5.73 (d, 2, 2-NH₂), 5.78 (s, 1,H-1), 6.83 (br s, 2, 6-NH₂).

B. 21-O-(2-methoxyethyl)-2,6-diaminopurine riboside and3′-O-(2-methoxyethyl)-2,6-diaminopurine riboside

To a solution of 2,6-diaminopurine riboside (10.0 g, 0.035 mol) in drydimethyl formamide (350 mL) at 0° C. under an argon atmosphere was addedsodium hydride (60% in oil, 1.6 g, 0.04 mol). After 30 min.,2-methoxyethyl bromide (0.44 mol) was added in one portion and thereaction was stirred at 25° C. for 16 h. Methanol (10 mL) was added andthe mixture was concentrated under reduced pressure to an oil (20 g).The crude product, containing a ratio of 4:1 of the 2′/3′ isomers, waschromatographed on silica gel (500 g, chloroform-methanol 4:1). Theappropriate fractions were combined and concentrated under reducedpressure to a semi-solid (12 g). This was triturated with methanol (50mL) to give a white, hygroscopic solid. The solid was dried at 40°C./0.2 mmHg for 6 h to give a pure 2′ product and the pure 3′ isomer,which were confirmed by NMR.

C. 3′-O-2-(methoxyethyl)guanosine

With rapid stirring, 3′-O-(2-methoxyethyl)-2,6-diaminopurine riboside(0.078 mol) was dissolved in monobasic sodium phosphate buffer (0.1 M,525 mL, pH 7.3-7.4) at 25° C. Adenosine deaminase (Sigma type II, 1unit/mg, 350 mg) was added and the reaction was stirred at 25° C. for 60h. The mixture was cooled to 5° C. and filtered. The solid was washedwith water (2×25 mL) and dried at 60° C./0.2 mmHg for 5 h to give 10.7 gof first crop material. A second crop was obtained by concentrating themother liquors under reduced pressure to 125 mL, cooling to 5° C.,collecting the solid, washing with cold water (2×20 mL) and drying asabove to give 6.7 g of additional material for a total of 15.4 g (31%from guanosine hydrate) of light tan solid; TLC purity 97%.

D. N²-Isobutyryl-3′-O-2-(methoxyethyl)guanosine

To a solution of3′-O-2-(methoxyethyl)guanosine(18.1 g, 0.0613 mol) inpyridine (300 mL) was added trimethyl silyl chloride (50.4 mL, 0.46mol). The reaction was stirred at 25° C. for-16 h. Isobutyryl chloride(33.2 mL, 0.316 mol) was added and the reaction was stirred for 4 h at25° C. The reaction was diluted with water (25 mL). After stirring for30 min, ammonium hydroxide (concentrated, 45 mL) was added until pH 6was reached. The mixture was stirred in a water bath for 30 min and thenevaporated under reduced pressure to an oil. The oil was suspended in amixture of ethyl acetate (600 mL) and water (100 mL) until a solutionformed. The solution was allowed to stand for 17 h at 25° C. Theresulting precipitate was collected, washed with ethyl acetate (2×50 mL)and dried at 60° C./0.2 mmHg for 5 h to give 16.1 g (85%) of tan solid;TLC purity 98%.

E. 5′-O-DMT)-N²-isobutyryl-3′-O-(2-methoxyethyl)guanosine

A solution of N²-Isobutyryl-3′-O-2-(methoxyethyl) guanosine (0.051 mol)in pyridine (150 mL) was evaporated under reduced pressure to dryness.The residue was redissolved in pyridine (300 mL) and cooled to 10-15° C.DMT-Cl (27.2 g, 95%, 0.080 mol) was added and the reaction was stirredat 25° C. for 16 h. The reaction was evaporated under reduced pressureto an oil, dissolved in a minimum of methylene chloride and applied on asilica gel column (500 g). The product was eluted with a gradient ofmethylene chloride-triethylamine (99:1) to methylenechloride-methanol-triethylamine (99:1:1). Selected fractions werecombined, concentrated under reduced pressure and dried at 40° C./0.2mmHg for 2 h to afford 15 g (15.5% from guanosine hydrate) of tan foam;TLC purity 98%.

EXAMPLE 8

[5′-O-(DMT)-N²-isobutyryl-3′-O-(2-methoxyethyl)guanosin-2′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite

The protected nucleoside from Example 7 (0.0486 mol) was placed in a dry1 L round bottom flask containing a Teflon stir-bar. The flask waspurged with argon. Anhydrous methylene chloride (400 mL) was cannulatedinto the flask to dissolve the nucleoside. Previously vacuum driedN,N-diisopropylaminohydrotetrazolide (3.0 g, 0.0174 mol) was added underargon. With stirring, bis-N,N-diisopropyl-aminocyanoethylphosphoramidite(18.8 g, 0.0689 mol) was added via syringe over 1 min (no exothermnoted). The reaction was stirred under argon at 25° C. for 16 h. Afterverifying the completion of the reaction by TLC, the reaction wastransferred to a separatory funnel (1 L). The reaction flask was rinsedwith methylene chloride (2×50 mL). The combined organic layer was washedwith saturated aq. sodium bicarbonate (200 mL) and then brine (200 mL).The organic layer was dried over sodium sulfate (50 g, powdered) for 2h. The solution was filtered and concentrated under reduced pressure toa viscous oil. The resulting phosphoramidite was purified by silica gelflash chromatography (800 g, ethyl acetate-triethylamine 99:1). Selectedwere combined, concentrated under reduced pressure, and dried at 25C./0.2 mmHg for 16 h to give 18.0 g (46%, 3% from guanosine hydrate) ofsolid foam TLC homogenous. ³¹P-NMR (CDCl₃, H₃PO₄ std.) δ 147.96; 148.20(diastereomers).

EXAMPLE 9

5′-O-DMT-3′-O-(2-methoxyethyl)-5-methyl-uridine-2′-O-succinate

5′-O-DMT-3′-O-(2-methoxyethyl)-thymidine was first succinylated on the2′-position. Thymidine nucleoside (4 mmol) was reacted with 10.2 mLdichloroethane, 615 mg (6.14 mmol) succinic anhydride, 570 μL (4.09mmol) triethylamine, and 251 mg (2.05 mmol) 4-dimethylaminopyridine. Thereactants were vortexed until dissolved and placed in heating block at55° C. for approximately 30 minutes. Completeness of reaction checked bythin layer chromatography (TLC). The reaction mixture was washed threetimes with cold 10% citric acid followed by three washes with water. Theorganic phase was removed and dried under sodium sulfate. Succinylatednucleoside was dried under P₂O₅ overnight in vacuum oven.

EXAMPLE 10

5′-O-DMT-3′-methoyethyl-5methyl-uridine-2-O-succinoyl Linked LCACPG5′-O-DMT-3′-

O-(2-methoxyethyl)-2′-O-succinyl-thymidine was coupled to controlledpore glass (CPG). 1.09 g (1.52 mmol) of the succinate were driedovernight in a vacuum oven along with 4-dimethylaminopyridine (DMAP),2,2′-dithiobis (5-nitro-pyridine) (dTNP), triphenylphosphine (TPP), andpre-acid washed CPG (controlled pore glass). After about 24 hours, DMAP(1.52 mmol, 186 mg) and acetonitrile (13.7 mL) were added to thesuccinate. The mixture was stirred under an atmosphere of argon using amagnetic stirrer. In a separate flask, dTNP (1.52 mmol, 472 mg) wasdissolved in acetonitrile (9.6 mL) and dichloromethane (4.1 mL) underargon. This reaction mixture was then added to the succinate. In anotherseparate flask, TPP (1.52 mmol, 399 mg) was dissolved in acetonitrile(37 mL) under argon. This mixture was then added to thesuccinate/DMAP/dTNP reaction mixture. Finally, 12.23 g pre-acid washedLCA CPG (loading=115.2 μmol/g) was added to the main reaction mixture,vortexed shortly and placed on shaker for approximately 3 hours. Themixture was removed from the shaker after 3 hours and the loading waschecked. A small sample of CPG was washed with copious amounts ofacetonitrile, dichloromethane, and then with ether. The initial loadingwas found to be 63 μmol/g (3.9 mg of CPG was cleaved withtrichloroacetic acid, the absorption of released trityl cation was readat 503 nm on a spectrophotometer to determine the loading.) The wholeCPG sample was then washed as described above and dried under P₂O₅overnight in vacuum oven. The following day, the CPG was capped with 25mL CAP A (tetrahydrofuran/acetic anhydride) and 25 mL CAP B(tetrahydrofuran/pyridine/1methyl imidazole) for approximately 3 hourson shaker. Filtered and washed with dichloromethane and ether. The CPGwas dried under P₂O₅ overnight in vacuum oven. After drying, 12.25 g ofCPG was isolated with a final loading of 90 μmol/g.

EXAMPLE 11

3′-O-Methoxyethyl-5-methyl-N-benzoyl-cytidine-2′-O-succinate

5′-O-DMT-3′-O-(2-methoxy)ethyl-N-benzoyl-cytidine was first succinylatedon the 2′-position. Cytidine nucleoside (4 mmol) was reacted with 10.2mL dichloroethane, 615 mg (6.14 mmol) succinic anhydride, 570 μL (4.09mmol) triethylamine, and 251 mg (2.05 mmol) 4-dimethylaminopyridine. Thereactants were vortexed until dissolved and placed in a heating block at55° C. for approximately 30 minutes. Completeness of reaction waschecked by thin layer chromatography (TLC). The reaction mixture waswashed three times with cold 10% citric acid followed by three washeswith water. The organic phase was removed and dried under sodiumsulfate. The succinylated nucleoside was dried under P₂O₅ overnight invacuum oven.

EXAMPLE 12

5′-O-DMT-3-O-methoxyethyl-5-methyl-N-benzoyl-cytidine-2′-O-succinoyllinked LCA CPG

5′-O-DMT-3′-O-(2-methoxyethyl)-2′-O-succinyl-N⁴-benzoyl cytidine wascoupled to controlled pore glass (CPG). 1.05 g (1.30 mmol) of thesuccinate were dried overnight in a vacuum oven along with4-dimethylaminopyridine (DMA), 2,2′-dithiobis (5-nitro-pyridine) (dTNP),triphenylphosphine (TPP), and pre-acid washed CPG (controlled poreglass). The following day, DMAP (1.30 mmol, 159 mg) and acetonitrile(11.7 mL) were added to the succinate. The mixture was “mixed” by amagnetic stirrer under argon. In a separate flask, dTNP (1.30 mmol, 400mg) was dissolved in acetonitrile (8.2 mL) and dichloromethane (3.5 mL)under argon. This reaction mixture was then added to the succinate. Inanother separate flask, TPP (1.30 mmol, 338 mg) was dissolved inacetonitrile (11.7 mL) under argon. This mixture was then added to thesuccinate/DMAP/dTNP reaction mixture. Finally, 10.46 g pre-acid washedLCA CPG (loading=115.2 μmol/g) were added to the main reaction mixture,vortexed shortly and placed on shaker for approximately 2 hours. Aportion was removed from shaker after 2 hours and the loading waschecked. A small sample of CPG was washed with copious amounts ofacetonitrile, dichloromethane, and then with ether. The initial loadingwas found to be 46 μmol/g. (3.4 mg of CPG were cleaved withtrichloroacetic acid). The absorption of released trityl cation was readat 503 nm on a spectrophotometer to determine the loading. The whole CPGsample was then washed as described above and dried under P₂O₅ overnightin vacuum oven. The following day, the CPG was capped with 25 mL CAP. A(tetrahydrofuran/acetic anhydride) and 25 mL CAP B(tetrahydrofuran/pyridine/1-methyl imidazole) for approximately 3 hourson a shaker. The material was filtered and washed with dichloromethaneand ether. The CPG was dried under P₂O₅ overnight in vacuum oven. Afterdrying, 10.77 g of CPG was isolated with a final loading of 63 μmol/g.

EXAMPLE 13

5′-O-DMT-3′-O-methoxyethyl-N6benzoyl-adenosine-2′-O-succinate

5′-O-DMT-3′-O-2-methoxyethyl)-N⁶-benzoyl adenosine was firstsuccinylated on the 2′-position. 3.0 g (4.09 mmol) of the adenosinenucleoside were reacted with 10.2 mL dichloroethane, 615 mg (6.14 mmol)succinic anhydride, 570 μL (4.09 mmol) triethylamine, and 251 mg (2.05mmol) 4-dimethylaminopyridine. The reactants were vortexed untildissolved and placed in heating block at 55° C. for approximately 30minutes. Completeness of reaction was checked by thin layerchromatography (TLC). The reaction mixture was washed three times withcold 10% citric acid followed by three washes with water. The organicphase was removed and dried under sodium sulfate. Succinylatednucleoside was dried under P₂O₅ overnight in vacuum oven.

EXAMPLE 14

5′-O-DMT-3′-O-(2-methoxyethyl)-N6benzoyl-adenosine-2′-O-succinoyl LinkedLCA CPG

Following succinylation,5′-O-DMT-3′-O-(2-methoxyethyl)-2′-O-succinyl-N⁶-benzoyl adenosine wascoupled to controlled pore glass (CPG). 3.41 g (4.10 mmol) of thesuccinate were dried overnight in a vacuum oven along with4-dimethylaminopyridine (DMAP), 2,2′-dithiobis (5-nitro-pyridine)(dTNP), triphenylphosphine (TPP), and pre-acid washed CPG (controlledpore glass). The following day, DMAP (4.10 mmol, 501 mg) andacetonitrile (37 mL) were added to the succinate. The mixture was“mixed” by a magnetic stirrer under argon. In a separate flask, dTNP(4.10 mmol, 1.27 g) was dissolved in acetonitrile (26 mL) anddichloromethane (11 mL) under argon. This reaction mixture was thenadded to the succinate. In another separate flask, TPP (4.10 mmol, 1.08g) was dissolved in acetonitrile (37 mL) under argon. This mixture wasthen added to the succinate/DMAP/dTNP reaction mixture. Finally, 33 gpre-acid washed LCA CPG (loading=115.2 μmol/g) were added to the mainreaction mixture, vortexed shortly and placed on shaker forapproximately 20 hours. Removed from shaker after 20 hours and theloading was checked. A small sample of CPG was washed with copiousamounts of acetonitrile, dichloromethane, and then with ether. Theinitial loading was found to be 49 μmol/g. (2.9 mg of CPG were cleavedwith trichloroacetic acid). The absorption of released trityl cation wasread at 503 nm on a spectrophotometer to determine the loading. Thewhole CPG sample was then washed as described above and dried under P₂O₅overnight in vacuum oven. The following day, the CPG was capped with 50mL CAP A (tetrahydrofuran/acetic anhydride) and 50 mL CAP B(tetrahydrofuran/pyridine/1-methyl imidazole) for approximately 1 houron the shaker. The material was filtered and washed with dichloromethaneand ether. The CPG was dried under P₂O₅ overnight in vacuum oven. Afterdrying, 33.00 g of CPG was obtained with a final loading of 66 μmol/g.

EXAMPLE 15

5′-O-DMT-3-O-(2-methoxyethyl)-N2-isobutyryl-guanosine-2′-O-succinate

5′-O-DMT-3′-O-(2-methoxyethyl)1-N²-isobutyryl guanosine was succinylatedon the 2′-sugar position. 3.0 g (4.20mmol)of the guanosine nucleosidewere reacted with 10.5 mL dichloroethane, 631 mg (6.30 mmol) succinicanhydride, 585 μL (4.20 mmol) triethylamine, and 257 mg (2.10 mmol)4-dimethylaminopyridine. The reactants were vortexed until dissolved andplaced in heating block at 55° C. for approximately 30 minutes.Completeness of reaction checked by thin layer chromatography (TLC). Thereaction mixture was washed three times with cold 10% citric acidfollowed by three washes with water. The organic phase was removed anddried under sodium sulfate. The succinylated nucleoside was dried underP₂O₅ overnight in vacuum oven.

EXAMPLE 16

5′-O-DMT-3′-O-methoxyethyl-N2-isobutyryl-guanosine2′-O-succinoyl LinkedLCA CPG

Following succinylation,5′-O-DMT-3′-O-(2-methoxyethyl)-2′-O-succinyl-N2-benzoyl guanosine wascoupled to controlled pore glass (CPG). 3.42 g (4.20 mmol) of thesuccinate were dried overnight in a vacuum oven along with4-dimethylaminopyridine (DMAP), 2,2′-dithiobis (5-nitro-pyridine)(dTNP), triphenylphosphine (TPP), and pre-acid washed CPG (controlledpore glass). The following day, DMAP (4.20 mmol, 513 mg) andacetonitrile (37.5 mL) were added to the succinate. The mixture was“mixed” by a magnetic stirrer under argon. In a separate flask, dTNP(4.20 mmol, 1.43 g) was dissolved in acetonitrile (26 mL) anddichloromethane (11 mL) under argon. This reaction mixture was thenadded to the succinate. In another separate flask, TPP (4.20 mmol, 1.10g) was dissolved in acetonitrile (37.5 mL) under argon. This mixture wasthen added to the succinate/DMAP/dTNP reaction mixture. Finally, 33.75 gpre-acid washed LCA CPG (loading=115.2 μmol/g) were added to the mainreaction mixture, vortexed shortly and placed on shaker forapproximately 20 hours. Removed from shaker after 20 hours and theloading was checked. A small sample of CPG was washed with copiousamounts of acetonitrile, dichloromethane, and then with ether. Theinitial loading was found to be 64 μmol/g. (3.4 mg of CPG were cleavedwith trichloroacetic acid). The absorption of released trityl cation wasread at 503 nm on a spectrophotometer to determine the loading. The CPGwas then washed as described above and dried under P₂O₅ overnight invacuum oven. The following day, the CPG was capped with 50 mL CAP A(tetrahydrofuran/acetic anhydride) and 50 mL CAP B(tetrahydrofuran/pyridine/1-methyl imidazole) for approximately 1 houron a shaker. The material was filtered and washed with dichloromethaneand ether. The CPG was dried under P₂O₅ overnight in vacuum oven. Afterdrying, 33.75 g. of CPG was isolated with a final loading of 72 μmol/g.

EXAMPLE 17

5′-O-DMT-3′-O-[hexyl-(6-phthalimido)]-uridine

2′,3′-O-Dibutyl stannylene-uridine was synthesized according to theprocedure of Wagner et. al., J. Org. Chem., 1974, 39,24. This compoundwas dried over P₂O₅ under vacuum for 12 hours. To a solution of thiscompound (29 g, 42.1 mmol) in 200 mL of anhydrous DMF were added (16.8g, 55 mmol) of 6-bromohexyl phthalimide and 4.5 g of sodium iodide andthe mixture was heated at 130° C. for 16 hours under argon. The reactionmixture was evaporated, co-evaporated once with toluene and the gummytar residue was applied on a silica column (500 g). The column waswashed with 2 L of EtOAc followed by eluting with 10% methanol(MeOH):90% EtOAc. The product, 2′- and 3′-isomers ofO-hexyl-6-N-phthalimido uridine, eluted as an inseparable mixture(R_(f)=0.64 in 10% MeOH in EtOAc). By ¹³C NMR, the isomeric ration wasabout 55% of the 2′ isomer and about 45% of the 3′ isomer. The combinedyield was 9.2 g (46.2%). This mixture was dried under vacuum andre-evaporated twice with pyridine. It was dissolved in 150 mL anhydrouspyridine and treated with 7.5 g of DMT-Cl (22.13 mmol) and 500 mg ofdimethylaminopyridine (DMAP). After 2 hours, thin layer chromatography(TLC; 6:4 EtOAc:Hexane) indicated complete disappearance of the startingmaterial and a good separation between 2′ and 3′ isomers (R_(f)=0.29 forthe 2′ isomer and 0.12 for the 3′ isomer). The reaction mixture wasquenched by the addition of 5 mL of CH₃OH and evaporated under reducedpressure. The residue was dissolved in 300 mL CH₂Cl₂, washedsuccessively with saturated NaHCO₃ followed by saturated NaCl solution.It was dried over Mg₂SO₄ and evaporated to give 15 g of a brown foamwhich was purified on a silica gel (500 g) to give 6.5 g of the2′-isomer and 3.5 g of the 3′ isomer.

EXAMPLE 18

5′-O-DMT-3′-O-[hexyl-(6-phthalimido)]-uridine-2′-O-(2-cyanoethyl-N,N,-diisopropyl)phosphoramidite

5′-DMT-3′-O-[hexyl-(6-phthalimido)]uridine (2 g, 2.6 mmol) was dissolvedin 20 mL anhydrous CH₂Cl₂. To this solution diisopropylaminotetrazolide(0.2 g, 1.16 mmol) and 2.0 mL 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphoramidite (6.3 mmol) were added with stirred overnight. TLC (1:1EtOAc/hexane) showed complete disappearance of starting material. Thereaction mixture was transferred with CH₂Cl₂ and washed with saturatedNaHCO₃ (100 mL), followed by saturated NaCl solution. The organic layerwas dried over anhydrous Na₂SO₄ and evaporated to yield 3.8 g of a crudeproduct, which was purified in a silica column (200 g) using 1:1hexane/EtOAc to give 1.9 g (1.95 mmol, 74% yield) of the desiredphosphoramidite.

EXAMPLE 19

Preparation of5′-O-DMT-3′-O-[hexyl-(6-phthalimido)]-uridine2′-O-succinoyl-aminopropylCPG

Succinylated and capped aminopropyl controlled pore glass (CPG; 500 Åpore diameter, aminopropyl CPG, 1.0 grams prepared according to Damhaet. al., Nucl Acids Res. 1990, 18, 3813.) was added to 12 mL anhydrouspyridine in a 100 mL round-bottom flask.1-(3-Dimethylaminopropyl)-3-ethyl-carbodiimide (DEC; 0.38 grams, 2.0mmol)], triethylamine (TEA; 100 μl, distilled over CaH₂),dimethylaminopyridine (DMAP; 0.012 grams, 0.1 mmol) and nucleoside5′-O-DMT-3′-O-[hexyl-(6-phthalimido)]uridine (0.6 grams, 0.77 mmol) wereadded under argon and the mixture shaken mechanically for 2 hours.Additional nucleoside (0.20 grams) was added and the mixture shaken for24 hours. The CPG was filtered off and washed successively withdichloromethane, triethylamine, and dichloromethane. The CPG was thendried under vacuum, suspended in 10 mL piperidine and shaken 15 minutes.The CPG was filtered off, washed thoroughly with dichloromethane andagain dried under vacuum. The extent of loading (determined byspectrophotometric assay of DMT cation in 0.3 M p-toluenesulfonic acidat 498 nm) was approximately 28 μmol/g. The5′-O-(DMT)-3′-O-[hexyl-(6-phthalimido]uridine-2′-O-succinoyl-aminopropylcontrolled pore glass was used to synthesize the oligomers in an ABI380B DNA synthesizer using phosphoramidite chemistry standardconditions. A four base oligomer 5′-GACU*-3′ was used to confirm thestructure of 3′-O-hexylamine tether introduced into the oligonucleotideby NMR. As expected a multiplet signal was observed between 1.0-1.8 ppmin ¹H NMR.

EXAMPLE 20

5′-O-DMT-3′-O-[hexylamino]-uridine

5′-O-(DMT)-3′-O-[hexyl-(6-phthalimido)]uridine (4.5 grams, 5.8 mmol) isdissolved in 200 mL methanol in a 500 mL flask. Hydrazine (1 ml, 31mmol) is added to the stirring reaction mixture. The mixture is heatedto 60-65° C. in an oil bath and refluxed 14 hours. The solvent isevaporated in vacuo and the residue is dissolved in dichloromethane (250mL) and extracted twice with an equal volume NH₄OH. The organic layer isevaporated to yield the crude product which NMR indicates is notcompletely pure. R_(f)=0 in 100% ethyl acetate. The product is used insubsequent reactions without further purification.

EXAMPLE 21

3′-O-[Propyl-(3-phthalimido)]-adenosine

To a solution of adenosine (20.0 g, 75 mmol) in dry dimethylformamide(550 ml) at 5° C. was added sodium hydride (60% oil, 4.5 g, 112 mmol).After one hour, N-(3-bromopropyl)-phthalimide (23.6 g, 86 mmol) wasadded and the temperature was raised to 30° C. and held for 16 hours.Ice is added and the solution evaporated in vacuo to a gum. The gum waspartitioned between water and ethyl acetate (4×300 mL). The organicphase was separated, dried, and evaporated in vacuo and the resultantgum chromatographed on silica gel (95/5 CH₂Cl₂/MeOH) to give a whitesolid (5.7 g) of the 2′-O-(propylphthalimide)adenosine. Thee fractionscontaining the 3′-O-(propylphthalimide)adenosine were chromatographed asecond time on silica gel using the same solvent system.

Crystallization of the 2′-O-(propylphthalimide)adenosine fractions frommethanol gave a crystalline solid, m.p. 123-124C. ¹H NMR (400 MHZ:DMSO-d₆) δ 1.70(m, 2H, CH₂), 3.4-3.7 (m, 6H, C_(5′), OCH₂, Phth CH₂),3.95 (q, 1H, C_(4′)H), 4.30 (q, 1H, C_(5′)H), 4.46 (t, 1H, C_(2′)H),5.15 (d, 1H, C_(3′)OH), 5.41 (t, 1H, C_(5′)OH), 5.95 (d, 1H, C_(1′)H)7.35 (s, 2H, NH₂), 7.8 (brs, 4H, Ar), 8.08 (s, 1H, C₂H) and 8.37 (s, 1H,C₈H). Anal. Calcd. C₂₁H₂₂N₆O₆: C, 55.03; H, 4.88; N, 18.49. Found: C,55.38; H, 4.85; N, 18.46.

Crystallization of the 3′-O-(propylphthalimide)adenosine fractions fromH₂O afforded an analytical sample, m.p. 178-179C. ¹H NMR (400 MHZ:DMSO-d₆) δ 5.86 (d, 1H, H-1′).

EXAMPLE 22

3′-O-[Propyl-(3-phthalimido)]-N6-benzoyl-adenosine

3′-O-(3-propylphthalimide)adenosine is treated with benzoyl chloride ina manner similar to the procedure of Gaffiey, et al., Tetrahedron Lett.1982, 23, 2257. Purification of crude material by chromatography onsilica gel (ethyl acetate-methanol) gives the title compound.

EXAMPLE 23

3′-O-[Propyl-(3-phthalimido)]-5′-O-DMT-N6-benzoyl-adenosine

To a solution of 3′-O-(propyl-3-phthalimide)-N⁶-benzoyladenosine (4.0 g)in pyridine (250 ml) is added DMT-Cl (3.3 g). The reaction is stirredfor 16 hours. The reaction is added to ice/water/ethyl acetate, theorganic layer separated, dried, and concentrated in vacuo and theresultant gum chromatographed on silica gel (ethyl acetate-methanoltriethylamine) to give the title compound.

EXAMPLE 24

3′-O-[Propyl-(3-phthalimido)]-51-O-DMT-N6-Benzoyl-adenosine-2′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite

3′-O-(Propyl-3-phthalimide)-5′-O-DMT-N⁶-benzoyladenosine is treated with(β-cyanoethoxy)chloro-N, N-diisopropyl)aminophosphane in a mannersimilar to the procedure of Seela, et al., Biochemistry 1987, 26, 2233.Chromatography on silica gel (EtOAc/hexane) gives the title compound asa white foam.

EXAMPLE 25

3′-O-(Aminopropyl)-adenosine

A solution of 3′-O-(propyl-3-phthalimide)adenosine (8.8 g, 19 mmol), 95%ethanol (400 mL) and hydrazine (10 mL, 32 mmol) is stirred for 16 hrs atroom temperature. The reaction mixture is filtered and filtrateconcentrated in vacuo. Water (150 mL) is added and acidified with aceticacid to pH 5.0. The aqueous solution is extracted with EtOAc (2×30 mL)and the aqueous phase is concentrated in vacuo to afford the titlecompound as a HOAc salt.

EXAMPLE 26

3′-O-[3-(N-trifluoroacetamido)propyl]-adenosine

A solution of 3′-O-(propylamino)adenosine in methanol (50 mL) andtriethylamine (15 mL, 108 mmol) is treated with ethyl trifluoroacetate(18 mL, 151 mmol). The reaction is stirred for 16 hrs and thenconcentrated in vacuo and the resultant gum chromatographed on silicagel (9/1, EtOAc/MeOH) to give the title compound.

EXAMPLE 27

N6-Dibenzoyl-3′-O-[3-(N-trifluoroacetamido)propyl]-adenosine

3′-O-[3-(N-trifluoroacetamido)propyl]adenosine is treated as per Example22 using a Jones modification wherein tetrabutylammonium fluoride isutilized in place of ammonia hydroxide in the work up. The crude productis purified using silica gel chromatography (EtOAc/MeOH 1/1) to give thetitle compound.

EXAMPLE 28

N6-Dibenzoyl-5′-O-DMT-3′-O-[3-(N-trifluoroacetamido)propyl]-adenosine

DMT-Cl (3.6 g, 10.0 mmol) is added to a solution ofN⁶-(dibenzoyl)-3′-O-[3-(N-trifluoro-acetamido)propyl)adenosine inpyridine (100 mL) at room temperature and stirred for 16 hrs. Thesolution is concentrated in vacuo and chromatographed on silica gel(EtOAc/TEA 99/1) to give the title compound.

EXAMPLE 29

N6-Dibenzoyl-5′-O-DMT-3′-O-[3-(N-trifluoroacetamido)propyl]-adenosine-2′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite

A solution ofN⁶-(dibenzoyl)-5′-O-(DMT)-3′-O-[3-(N-trifluoroacetamido)propyl]-adenosinein dry CH₂Cl₂ is treated with bis-N,N-diisopropylamino cyanoethylphosphite (1.1 eqiv) and N,N-diisopropylaminotetrazolide (catalyticamount) at room temperature for 16 hrs. The reaction is concentrated invacuo and chromatographed on silica gel (EtOAc/hexane/TEA 6/4/1) to givethe title compound.

EXAMPLE 30

3′-O-(butylphthalimido)-adenosine

The title compound is prepared as per Example 21, usingN-(4-bromobutyl)phthalimide in place of the 1-bromopropane.Chromatography on silica gel (EtOAC-MeOH) gives the title compound. ¹HNMR (200 MHZ, DMSO-d₆) δ 5.88 (d, 1H, C₁H).

EXAMPLE 31

N6-Benzoyl-3′-O-(butylphthalimido)-adenosine

Benzoylation of 3′-O-(butylphthalimide)adenosine as per Example 22 givesthe title compound.

EXAMPLE 32

N6-Benzoyl-5′-O-DMT-3′-O-(butylphthalimido)-adenosine

The title compound is prepared from3′-O-(butyl-phthalimide)-N⁶-benzoyladenosine as per Example 22.

EXAMPLE 33

N6-Benzoyl-5′-O-DMT-3′-O-(butylphthalimido)-Adenosine-2′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite

The title compound is prepared from3′-O-(butylphthalimide)-5′-O-DMT-N⁶-benzoyladenosine as per Example 24.

EXAMPLE 34

3′-O-(Pentylphthalimido)-adenosine

The title compound is prepared as per Example 21, usingN-(5-bromopentyl)phthalimide. The crude material from the extraction ischromatographed on silica gel using CHCl₃/MeOH (95/5) to give a mixtureof the 2′ and 3′ isomers. The 2′ isomer is recrystallized from EtOH/MeOH8/2. The mother liquor is rechromatographed on silica gel to afford the3′ isomer. 2′-O-(Pentylphthalimido)adenosine: M.P. 159-160° C. Anal.Calcd. for C₂₃H₂₄N₆O₅: C, 57.26; H, 5.43; N, 17.42. Found: C, 57.03; H,5.46; N, 17.33. 3′-O-(Pentylphthalimido)adenosine: ¹H NMR (DMSO-d₆) δ5.87 (d, 1H, H-1′).

EXAMPLE 35

N6-Benzoyl-3′-O-(pentylphthalimido)-adenosine

Benzoylation of 3′-O-(pentylphthalimido)adenosine is achieved as per theprocedure of Example 22 to give the title compound.

EXAMPLE 36

N6Benzoyl-5′-O-DMT-3′-O-(pentylphthalimido)-adenosine

The title compound is prepared from3′-O-(pentyl-phthalimide)-N⁶-benzoyladenosine as per the procedure ofExample 23. Chromatography on silica gel (ethylacetate, hexane,triethylamine), gives the title compound.

EXAMPLE 37

N6-Benzoyl-5′-O-DMT-31-O-(pentylphthalimido)-adenosine-2′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite

The title compound is prepared from3′-O-(pentyl-phthalimide)-5′-O-(DMT)-N⁶-benzoyladenosine as per theprocedure of Example 24 to give the title compound.

EXAMPLE 38

3′-O-(Propylphthalimido)uridine

A solution of uridine-tin complex (48.2 g, 115 mmol) in dry DM (150 ml)and N-(3-bromopropyl)phthalimide (46 g, 172 mmol) was heated at 130° C.for 6 hrs. The crude product was chromatographed directly on silica gelCHCl₃/MeOH 95/5. The isomer ratio of the purified mixture was 2′/3′81/19. The 2′ isomer was recovered by crystallization from MeOH. Thefiltrate was rechromatographed on silica gel using CHCl₃CHCl₃/MeOH(95/5) gave the 3′ isomer as a foam. 2′-O-(Propylphthalimide)uridine:Analytical sample recrystallized from MeOH, m.p. 165.5-166.5C, ¹H NMR(200 MHZ, DMSO-d₆) δ 1.87 (m, 2H, CH₂), 3.49-3.65 (m, 4H, C_(2′)H),3.80-3.90 (m, 2H, C_(3′)H₁C_(4′)H), 4.09(m, 1H, C_(2′)H), 5.07 (d, 1h,C_(3′)OH), 5.16 (m, 1H, C_(5′)OH), 5.64 (d, 1H, CH═), 7.84 (d, 1H,C_(1′)H), 7.92 (bs, 4H, Ar), 7.95 (d, 1H, CH═) and 11.33 (s, 1H, ArNH).Anal. C₂₀H₂₁N₃H₈, Calcd. C, 55.69; H, 4.91; N, 9.74. Found, C, 55.75; H,5.12; N, 10.01. 3′-O-(Propylphthalimide)uridine: ¹H NMR (DMSO-d₆) δ 5.74(d, 1H, H-1′).

EXAMPLE 39

3′-O-(Aminopropyl)-uridine

The title compound is prepared as per the procedure of Example 25.

EXAMPLE 40

3′-O-[3-(N-trifluoroacetamido)propyl]-uridine

3′-O-(Propylamino)uridine is treated as per the procedure of Example 26to give the title compound.

EXAMPLE 41

5′-O-DMT-3′-O-[3-(N-trifluoroacetamido)propyl]-uridine

3′-O-[3-(N-trifluoroacetamido)propyl]uridine is treated as per theprocedure of Example 28 to give the title compound.

EXAMPLE 42

5′-O-DMT-3′-O-[3-(N-trifluoroacetamido)propyl]-uridine-2′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite

5′-O-(DMT)-3′-O-[3-(N-trifluoroacetamido)propyl]uridine is treated asper the procedure of Example 29 to give the title compound.

EXAMPLE 43

3′-O-(Propylphthalimido)-cytidine

The title compounds were prepared as per the procedure of Example 21.2′-O-(propylphthalimide)cytidine: ¹H NMR (200 MHZ, DMSO-d₆) δ 5.82 (d,1H, C_(1′)H). 3′-O-(propylphthalimide)cytidine: ¹H NMR (200 MHZ,DMSO-d₆) δ 5.72 (d, 1H, C_(1′)H).

EXAMPLE 44

3′-O-(Aminopropyl)-cytidine

3′-O-(Propylphthalimide)cytidine is treated as per the procedure ofExample 25 to give the title compound.

EXAMPLE 45

3′-O-[3-(N-trifluoroacetamido)propyl]-cytidine

3′-O-(Propylamino)cytidine is treated as per the procedure of Example 26to give the title compound.

EXAMPLE 46

N4-Benzoyl-3′-O-[3-(N-trifluoroacetamido)propyl]-cytidine

3′-O-[3-(N-trifluoroacetamido)propyl]cytidine is treated as per theprocedure of Example 27 to give the title compound.

EXAMPLE 47

N4-Benzoyl-5′-O-DMT-3′-O-[3-(N-trifluoroacetamido)propyl]-cytidine

N⁴-(Benzoyl)-3′-O-[3-(N-trifluoroacetamido)propyl]cytidine is treated asper the procedure of Example 28 to give the title compound.

EXAMPLE 48

N4-Benzoyl-5′-O-DMT-3′-O-[3-(N-trifuoroacetamido)propyl]-cytidine-2′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite

N⁴-(Benzoyl)-5′-O-(DMT)-3′-O-[3-(N-trifluoroacetamido)propyl]cytidine istreated as per the procedure of Example 29 to give the title compound.

EXAMPLE 49

General Procedures for Oligonucleotide Synthesis

Oligonucleotides were synthesized on a Perseptive Biosystems Expedite8901 Nucleic Acid Synthesis System. Multiple 1-μmol syntheses wereperformed for each oligonucleotide. Trityl groups were removed withtrichloroacetic acid (975 μL over one minute) followed by anacetonitrile wash. All standard amidites (0.1 M) were coupled twice percycle (total coupling time was approximately 4 minutes). All novelamidites were dissolved in dry acetonitrile (100 mg of amidite/1 mLacetonitrile) to give approximately 0.08-0.1 M solutions. Total couplingtime was approximately 6 minutes (105 μL of amidite delivered).1-H-tetrazole in acetonitrile was used as the activating agent. Excessamidite was washed away with acetonitrile.(1S)-(+)-(10-camphorsulfonyl)oxaziridine (CSO, 1.0 g CSO/8.72 mL dryacetonitrile) was used to oxidize (4 minute wait step)phosphodiesterlinkages while 3H-1,2-benzodithiole-3-one-1,1-dioxide (Beaucage reagent,3.4 g Beaucage reagent/200 mL acetonitrile) was used to oxidize (oneminute wait step) phosphorothioate linkages. Unreacted functionalitieswere capped with a 50:50 mixture of tetrahydrofuran/acetic anhydride andtetrahydrofuran/pyridine/1-methyl imidazole. Trityl yields were followedby the trityl monitor during the duration of the synthesis. The finalDMT group was left intact. The oligonucleotides were deprotected in 1 mL28.0-30% ammonium hydroxide (NH₄OH) for approximately 16 hours at 55° C.Oligonucleotides were also made on a larger scale (20 μmol/synthesis).Trityl groups were removed with just over 8 mL of trichloroacetic acid.All standard amidites (0.1M) were coupled twice per cycle (13 minutecoupling step). All novel amidites were also coupled four times percycle but the coupling time was increased to approximately 20 minutes(delivering 480 μL of amidite). Oxidation times remained the same butthe delivery of oxidizing agent increased to approximately 1.88 mL percycle. Oligonucleotides were cleaved and deprotected in 5 mL 28.0-30%NH₄OH at 55° C., for approximately 16 hours.

TABLE I 3′-O-(2-methoxyethyl) containing 2′-5′ linked o-ligonucleotides. SEQ ID NO. (ISIS) Back- Chem- # # Sequence (5′ 3′)¹bone istry 4 (17176) ATG-CAT-TCT-GCC-CCC-AAG- P = S 3′-O-MOE GA* 5(17177) ATG-CAT-TCT-GCC-CCC-AAG- P = S 3′-O-MOE G*A* 6 (17178)ATG-CAT-TCT-GCC-CCC- P = S/ 3′-O-MOE AAG_(O)-G* _(O) A* P = O 7 (17179)A*TG-CAT-TCT-GCC-CCC- P = S 3′-O-MOE AAG-GA* 8 (17180)A*TG-CAT-TCT-GCC-CCC- P = S 3′-O-MOE AAG-G*A* 9 (17181) A*_(O)TG-CAT-TCT-GCC-AAA- P = S/ 3′-O-MOE AAG_(O)-G* _(O) A* P = O 10(21415) A*T*G-CAT-TCT-GCC-AAA- P = S 3′-O-MOE AAG-G*A* 11 (21416) A*_(O) T* _(O)G-CAT-TCT-GCC-AAA- P = S/ 3′-O-MOE AAG_(O)-G* _(O) A* P = O(21945) A*A*A* P = O 3′-O-MOE (21663) A*A*A*A* P = O 3′-O-MOE (20389)A*U*C*G* P = O 3′-O-MOE 12 (20390) C*G*C*-G*A*A*-T*T*C*-G* P = O3′-O-MOE C*G* ¹All nucleosides with an asterisk contain3′-O-(2-methoxyethyl).

EXAMPLE 50 General Procedure for Purification of Oligonucleotides

Following cleavage and deprotection step, the crude oligonucleotides(such as those synthesized in Example 49) were filtered from CPG usingGelman 0.45 μm nylon acrodisc syringe filters. Excess NH₄OH wasevaporated away in a Savant AS160 automatic speed vac. The crude yieldwas measured on a Hewlett Packard 8452A Diode Array Spectrophotometer at260 nm. Crude samples were then analyzed by mass spectrometry (MS) on aHewlett Packard electrospray mass spectrometer and by capillary gelelectrophoresis (CGE) on a Beckmann P/ACE system 5000. Trityl-onoligonucleotides were purified by reverse phase preparative highperformance liquid chromatography (HPLC). HPLC conditions were asfollows: Waters 600E 991 detector; Waters Delta Pak C4 column (7.8×300mm); Solvent A: 50 mM triethylammonium acetate (TEA-Ac), pH 7.0; B: 100%acetonitrile; 2.5 mL/min flow rate; Gradient: 5% B for first fiveminutes with linear increase in B to 60% during the next 55 minutes.Larger oligo yields from the larger 20 μmol syntheses were purified onlarger HPLC columns (Waters Bondapak HC18HA) and the flow rate wasincreased to 5.0 mL/min. Appropriate fractions were collected andsolvent was dried down in speed vac. Oligonucleotides were detritylatedin 80% acetic acid for approximately 45 minutes and lyophilized again.Free trityl and excess salt were removed by passing detritylatedoligonucleotides through Sephadex G-25 (size exclusion chromatography)and collecting appropriate samples through a Pharmacia fractioncollector. Solvent again evaporated away in speed vac. Purifiedoligonucleotides were then analyzed for purity by CGE, HPLC (flow rate:1.5 mL/min; Waters Delta Pak C4 column, 3.9×300mm), and MS. The finalyield was determined by spectrophotometer at 260 nm.

TABLE II Physical characteristics of 3'-O-(2-methoxyethyl) containing2'-5' linked oligonucleotides. HPLC² Expected Observed T_(R) PurifiedMass Mass (min.) #Ods(260 nm) 17176 6440.743 6440.300 23.47 3006 171776514.814 6513.910 23.67 3330 17178 6482.814 6480.900 23.06 390 171796513.798 6513.560 23.20 3240 17180 6588.879 6588.200 23.96 3222 171816540.879 6539.930 23.01 21415 6662.976 6662.700 24.18 4008 214166598.969 6597.800 23.01 3060 21945 1099.924 1099.300 19.92 121 216631487.324 1486.800 20.16 71 20389 1483.000 1482.000 62 20390 4588.0004591.000 151 ²Conditions: Waters 600E with detector 991; Waters C4column (3.9 × 300 mm); Solvent A: 50 mM TEA-Ac, pH 7.0; B: 100%acetonitrile; 1.5 mL/min. flow rate; Gradient: 5% B for first fiveminutes with linear increase in B to 60% during the next 55 minutes.T_(m) Studies on Modified Oligonucleotides

Oligonucleotides synthesized in Examples 49 and 50 were evaluated fortheir relative ability to bind to their complementary nucleic acids bymeasurement of their melting temperature (T_(m)). The meltingtemperature (T_(m)), a characteristic physical property of doublehelices, denotes the temperature (in degrees centigrade) at which 50%helical (hybridized) versus coil (unhybridized) forms are present. T_(m)is measured by using the UV spectrum to determine the formation andbreakdown (melting) of the hybridization complex. Base stacking, whichoccurs during hybridization, is accompanied by a reduction in UVabsorption (hypochromicity). Consequently, a reduction in UV absorptionindicates a higher T_(m). The higher the T_(m), the greater the strengthof the bonds between the strands.

Selected test oligonucleotides and their complementary nucleic acidswere incubated at a standard concentration of 4 μM for eacholigonucleotide in buffer (100 mM NaCl, 10 mM sodium phosphate, pH 7.0,0.1 mM EDTA). Samples were heated to 90 ° C. and the initial absorbancetaken using a Guilford Response II Spectrophotometer (Coming). Sampleswere then slowly cooled to 15° C. and then the change in absorbance at260 nm was monitored with heating during the heat denaturationprocedure. The temperature was increased by 1 degree ° C./absorbancereading and the denaturation profile analyzed by taking the 1^(st)derivative of the melting curve. Data was also analyzed using atwo-state linear regression analysis to determine the Tm=s. The resultsof these tests for the some of the oligonucleotides from Examples 49 and50 are shown in Table III below.

TABLE III Tm Analysis of Oligonucleotides SEQ # ID: Back- Mods # NO.(ISIS) bone Link- 2′- # # Sequence (5′-3′) T_(m)8 ages 5′ 13 (11061)ATG-CAT-TCT-GCC- P = S 61.4 0 0 CCC-AAG-GA 4 (17176) ATG-CAT-TCT-GCC- P= S 61.4 1 0 CCC-AAG-GA* 5 (17177) ATG-CAT-TCT-GCC- P = S 61.3 2 1CCC-AAG-G*A* 6 (17178) ATG-CAT-TCT-GCC- P = S/ 61.8 2 1 CCC-AAG_(O)-G*_(O) A* P = O 7 (17179) A*TG-CAT-TCT-GCC- P = S 61.1 2 1 CCC-AAG-GA* 8(17180) A*TG-CAT-TCT-GCC- P = S 61.0 3 2 CCC-AAG-G*A* 9 (17181) A*_(O)TG-CAT-TCT-GCC- P = S/ 61.8 3 2 AAA-AAG_(O)-G* _(O) A* P = O 10(21415) A*T*G-CAT-TCT-GCC- P = S 61.4 4 3 AAA-AAG-G*A* 11 (21416) A*_(O) T* _(O)G-CAT-TCT- P = S/ 61.7 4 3 GCC-AAA-AAG_(O)-G* _(O) A* P = O

EXAMPLE 52

NMR Experiments on Modified Oligonucleotides Comparison of 3′,5′ Versus2′,5′ Internucleotide Linkages and 2′-substituents Versus3′-substituents by NMR

The 400 MHz ¹H spectrum of oligomer d(GAU₂*CT), whereU₂*=2′-O-aminohexyluridine showed 8 signals between 7.5 and 9.0 ppmcorresponding to the 8 aromatic protons. In addition, the anomericproton of U* appears as a doublet at 5.9 ppm with J₁′,₂′=7.5 Hz,indicative of C2′-endo sugar puckering. The corresponding 2′-5′ linkedisomer shows a similar structure with J₁′,₂′=3.85 Hz at 5.75 ppm,indicating an RNA type sugar puckering at the novel modification sitefavorable for hybridization to an mRNA target. The proton spectrum ofthe oligomer d(GACU₃*), where U₃*=3′-O-hexylamine, showed the expected 7aromatic proton signals between 7.5 and 9.0 ppm and the anomeric protondoublet at 5.9 ppm with J₁′,₂′=4.4 Hz. This suggests more of a C3′-endopuckering for the 3′-O-alkylamino compounds compared to their 2′analogs. ³¹P NMR of these oligonucleotides showed the expected 4 and 3signals from the internucleotide phosphate linkages for d(GAU*CT) andd(GACU*), respectively. 3′-5′ Linked vs. 2′-5′ linked have differentretention times in RP HPLC and hence different lipophilicities, implyingpotentially different extent of interactions with cell membranes.

EXAMPLE 53

T_(m) Analysis of 2′,5′-linked Oligonucleotides Versus 3′,5′-linkedOligonucleotides

Thermal melts were done as per standarized literature procedures.Oligonucleotide identity is as follows:

Oligonucleotide A is a normal 3′-5′ linked phosphodiesteroligodeoxyribonucleotide of the sequence d(GGC TGU* CTG CG) SEQ ID NO:14 where the * indicates the attachment site of a 2′-aminolinker.Oligonucleotide B is a normal 3′-5′ linked phosphodiesteroligoribonucleotide of the sequence d(GGC TGU* CTG CG) SEQ ID NO: 14where the * indicates the attachment site of a 2′-aminolinker. Each ofthe ribonucleotides of the oligonucleotide, except the one bearing the *substituent, are 2′-O-methyl ribonucleotides. Oligonucleotide C has2′-5′ linkage at the * position in addition to a 3′-aminolinker at thissite. The remainder of the oligonucleotide is a phosphodiesteroligodeoxyribonucleotide of the sequence d(GGC TGU* CTG CG) SEQ ID NO:14. The base oligonucleotide (no 2′-aminolinker) was not included in thestudy.

TABLE IIIa DNA RNA OLIGONUCLEOTIDE MODIFICATION TARGET TARGET A none52.2 54.1 2'-aminolinker 50.9 55.5 B none 51.5 72.3 2'-aminolinker 49.869.3 C none NA 3'-aminolinker 42.7 51.7

The 2′-5′ linkages demonstrated a higher melting temperature against anRNA target compared to a DNA target.

EXAMPLE 54

Snake Venom Phosphodiesterase and Liver Homogenate Experiments onOligonucleotide Stability

The following oligonucleotides were synthesized following the procedureof Example 49.

TABLE IV Modified Oligonucleotides synthesized to evaluate stability SEQID NO. (ISIS) Back- Che- # # Sequence (5′-3′) bone mistry 15 (22110)TTT-TTT-TTT-TTT-TTT-T*T* P = O 3′-O-MOE T*-T* 16 (22111)TTT-TTT-TTT-TTT-TTT-T ^(#) T ^(#) P = O 3′-O-MOE T ^(#)-U ^(#) 15(22112) TTT-TTT-TTT-TTT-TTT-T*T* P = S 3′-O-MOE T*-T* 16 (22113)TTT-TTT-TTT-TTT-TTT-T ^(#) T ^(#) P = S 3′-O-MOE T ^(#)-U ^(#) 15(22114) TTT-TTT-TTT-TTT-TTT_(O)-T* P = S/ 3′-O-MOE _(O) T* _(O) T* _(O)T* P = O 16 (22115) TTT-TTT-TTT-TTT-TTT_(O)-T ^(#) P = S/ 3′-O-MOE _(O)T ^(#) _(O) T ^(#) _(O)-U ^(#) P = O ¹All nucleosides with an asteriskcontain 3′-O-(2-methoxyethyl). All nucleosides with a # contain2′-O-(2-methoxyethyl).The oligonucleotides were purified following the procedure of Example 50and analyzed for their structure.

TABLE V Properties of Modified Oligonucleotides #Ods(260 nm) SEQ IDExpected Observed HPLC² Purified NO.# (ISIS)# #Sequence (5′-3′)¹ MassMass T_(R) (min.) 15 (22110) TTT-TTT-TTT-TTT-TTT-T*T*T*-T* 6314.1896315.880 20.39 174 16 (22111) TTT-TTT-TTT-TTT-TTT-T ^(#) T ^(#) T ^(#)-U^(#) 6004.777 5997.490 20.89 147 15 (22112)TTT-TTT-TTT-TTT-TTT-T*T*T*-T* 6298.799 6301.730 25.92  224⁻ 16 (22113)TTT-TTT-TTT-TTT-TTT-T ^(#) T ^(#) T ^(#)-U ^(#) 6288.745 6286.940 24.77209 15 (22114) TTT-TTT-TTT-TTT-TTT_(O)-T* _(O) T* _(O) T* _(O)-T*6234.799 6237.150 24.84 196 16 (22115) TTT-TTT-TTT-TTT-TTT_(O)-T ^(#)_(O) T ^(#) _(O) T ^(#) _(O)-U ^(#) 6224.745 6223.780 23.30 340 ¹A11nucleosides with an asterisk contain 3′-O-(2-methoxyethyl). Allnucleosides with a # contain 2′-O-(2-methoxy) ethyl. ²Conditions: Waters600E with detector 991; Waters C4 column (3.9 × 300 mm); Solvent A: 50mM TEA-Ac, pH 7.0; B: 100% acetonitrile; 1.5 mL/min. flow rate;Gradient: 5% B for first five minutes with linear increase in B to 60%during the next 55 minutes.

EXAMPLE 55

3′-O-Aminopropyl Modified Oligonucleotides

Following the procedures illustrated above for the synthesis ofoligonucleotides, modified 3′-amidites were used in addition toconventional amidites to prepare the oligonucleotides listed in tablesVI and VII. Nucleosides used include:N6-benzoyl-3′-O-propylphthalimido-A-2′-amidite,2′-O-propylphthaloyl-A-3′-amidite,2′-O-methoxyethyl-thymidine-3′-amidite (RIC, Inc.),2′-O-MOE-G-3′-amidite (RI Chemical),2′-O-methoxyethyl-5-methylcytidine-3′-amidite,2-O-methoxyethyl-adenosine-3′-amidite (RI Chemical), and5-methylcytidine-3′-amidite. 3′-propylphthalimido-A and2′-propylphthalimido-A were used as the LCA-CPG solid support. Therequired amounts of the amidites were placed in dried vials, dissolvedin acetonitrile (unmodified nucleosides were made into 1M solutions andmodified nucleosides were 100 mg/mL), and connected to the appropriateports on a Millipore Expedite™ Nucleic Acid Synthesis System. Solidsupport resin (60 mg) was used in each column for 2×1 μmole scalesynthesis (2 columns for each oligo were used). The synthesis was runusing the IBP-PS(1 μmole) coupling protocol for phosphorothioatebackbones and CSO-8 for phosphodiesters. The trityl reports indicatednormal coupling results.

After synthesis the oligonucleotides were deprotected with conc.ammonium hydroxide(aq) containing 10% of a solution of 40% methylamine(aq) at 55° C. for approximately 16 hrs. Then they were evaporated,using a Savant AS160 Automatic SpeedVac, (to remove ammonia) andfiltered to remove the CPG-resin. The crude samples were analyzed by MS,HPLC, and CE. Then they were purified on a Waters 600E HPLC system witha 991 detector using a Waters C4 Prep. scale column (Alice C4 Prep.) andthe following solvents: A: 50 mM TEA-Ac, pH 7.0 and B: acetonitrileutilizing the AMPREP2@ method. After purification the oligonucleotideswere evaporated to dryness and then detritylated with 80% acetic acid atroom temp. for approximately 30 min. Then they were evaporated. Theoligonucleotides were dissolved in conc. ammonium hydroxide and runthrough a column containing Sephadex G-25 using water as the solvent anda Pharmacia LKB SuperFrac fraction collector. The resulting purifiedoligonucleotides were evaporated and analyzed by MS, CE, and HPLC.

TABLE VI Oligonucleotides bearing Aminopropyl Substituents SEQ ID NO.(ISIS) Back- # # Sequence (5′-3′)¹ bone 19 (23185-1)A*TG-CAT-TCT-GCC-CCC-AAG-GA* P = S 19 (23186-1)A*TG-CAT-TCT-GCC-CCC-AAG-G A* P = S 20 (23187-1) A*_(OT) _(O) G _(O)-C_(O) A _(S)T_(S)-T_(S)C_(S)T_(S)-G_(S)C_(S)C_(S)-C_(S) P = S/C_(S)C_(S)-A _(O) A _(O) G _(O)-G _(O) A* P = O 20 (23980-1) A*_(O) T_(O) G _(O)-C _(O) A _(S)T_(s)-T_(S)C_(S)T_(S)-G_(S)C_(S)C_(S)-C_(S) P= S/ C_(S)C_(S)-A _(O) A _(O) G _(O)-G _(O) A* P = O 19 (23981-1)A*TG-CAT-TCT-GCC-CCC-AAG-G A* P = S 19 (23982-1)A*TG-CAT-TCT-GCC-CCC-AAG-GA* P = S ¹All underlined nucleosides bear a2′-O-methoxyethyl substituent; internucleotide linkages in PS/POoligonucleotides are indicated by subscript >s= and >o= notationsrespectively; A* = 3′-aminopropyl-A; A* = 2′-aminopropyl-A; C= 5-methyl-C

TABLE VII Aminopropyl Modified Oligonucleotides HPLC CE Re- ExpectedObserved Retention tention Crude Final Mass Mass Time Time Yield YieldISIS # (g/mol) (g/mol) (min) (min) (Ods) (Ods) 23185-1 6612.065 6610.523.19 5.98 948 478 23186-1 7204.697 7203.1 24.99 6.18 1075 379 23187-17076.697 7072.33 23.36 7.56 838 546 23980-1 7076.697 7102.31 23.42 7.16984 373 23981-1 7204.697 7230.14 25.36 7.18 1170 526 23982-1 6612.0656635.71 23.47 7.31 1083 463

EXAMPLE 56

In vivo Stability of Modified Oligonucleotides

The in vivo stability of selected modified oligonucleotides synthesizedin Examples 49 and 55 was determined in BALB/c mice. Following a singlei.v. administration of 5 mg/kg of oligonucleotide, blood samples weredrawn at various time intervals and analyzed by CGE. For eacholigonucleotide tested, 9 male BALB/c mice (Charles River, Wilmington,Mass.) weighing about 25 g were used. Following a one weekacclimatization the mice received a single tail-vein injection ofoligonucleotide (5 mg/kg) administered in phosphate buffered saline(PBS), pH 7.0. One retro-orbital bleed (either at 0.25, 0.5, 2 or 4 hpost-dose) and a terminal bleed (either 1, 3, 8, or 24 h post-dose) werecollected from each group. The terminal bleed (approximately 0.6-0.8 mL)was collected by cardiac puncture following ketamine/xylazineanasthesia. The blood was transferred to an EDTA-coated collection tubeand centrifuged to obtain plasma. At termination, the liver and kidneyswere collected from each mouse. Plasma and tissue homogenates were usedfor analysis to determine intact oligonucleotide content by CGE. Allsamples were immediately frozen on dry ice after collection and storedat −80C until analysis.

The CGE analysis indicated the relative nuclease resistance of2′,5′-linked oligomers compared to ISIS 11061 (Table III, Example 51)(uniformly 2′-deoxy-phosphorothioate oligonucleotide targeted to mousec-raf). Because of the nuclease resistance of the 2′,5′-linkage, coupledwith the fact that 3′-methoxyethoxy substituents are present and affordfurther nuclease protection the oligonucleotides ISIS 17176, ISIS 17177,ISIS 17178, ISIS 17180, ISIS 17181 and ISIS 21415 were found to be morestable in plasma, while ISIS 11061 (Table III) was not. Similarobservations were noted in kidney and liver tissue. This implies that2′,5′-linkages with 3′-methoxyethoxy substituents offer excellentnuclease resistance in plasma, kidney and liver against 5′-exonucleasesand 3′-exonucleases. Thus oligonucleotides with longer durations ofaction can be designed by incorporating both the 2′,5′-linkage and3′-methoxyethoxy motifs into their structure. It was also observed that2′,5′-phosphorothioate linkages are more stable than2′,5′-phosphodiester linkages. A plot of the percentage of full lengtholigonucleotide remaining intact in plasma one hour followingadministration of an i.v. bolus of 5 mg/kg oligonucleotide is shown inFIG. 4.

A plot of the percentage of full length oligonucleotide remaining intactin tissue 24 hours following administration of an i.v. bolus of 5 mg/kgoligonucleotide is shown in FIG. 5.

CGE traces of test oligonucleotides and a standard phosphorothioateoligonucleotide in both mouse liver samples and mouse kidney samplesafter 24 hours are shown in FIG. 6. As is evident from these traces,there is a greater amount of intact oliogonucleotide for theoligonucleotides of the invention as compared to the standard seen inpanel A. The oligonucleotide shown in panel B included one substituentof the invention at each of the 5′ and 3′ ends of a phosphorothioateoligonucleotide while the phosphorothioate oligonucleotide seen in panelC included one substituent at the 5′ end and two at the 3′ end. Theoligonucleotide of panel D includes a substituent of the inventionincorporated in a 2′,5′ phosphodiester linkage at both its 5′ and 3′ends. While less stable than the oligonucleotide seen in panels B and C,it is more stable than the full phosphorothioate standardoligonucleotide of panel A.

EXAMPLE 57

Control of c-raf Message in bEND Cells Using Modified Oligonucleotides

In order to assess the activity of some of the oligonucleotides, an invitro cell culture assay was used that measures the cellular levels ofc-raf expression in bEND cells.

Cells and Reagents

The bEnd.3 cell line, a brain endothelioma, was obtained from Dr. WernerRisau (Max-Planck Institute). Opti-MEM, trypsin-EDTA and DMEM with highglucose were purchased from Gibco-BRL (Grand Island, N.Y.). Dulbecco'sPBS was purchased from Irvine Scientific (Irvine, Calif.). Sterile, 12well tissue culture plates and Facsflow solution were purchased fromBecton Dickinson (Mansfield, Mass.). Ultrapure formaldehyde waspurchased from Polysciences (Warrington, Pa.). NAP-5 columns werepurchased from Pharmacia (Uppsala, Sweden).

Oligonucleotide Treatment

Cells were grown to approximately 75% confluency in 12 well plates withDMEM containing 4.5 g/L glucose and 10% FBS. Cells were washed 3 timeswith Opti-MEM pre-warmed warmed to 37° C. Oligonucleotide were premixedwith a cationic lipid (Lipofectin reagent, (GIBCO/BRL) and, seriallydiluted to desired concentrations and transferred on to washed cells fora 4 hour incubation at 37° C. Media was then removed and replaced withnormal growth media for 24 hours for northern blot analysis of mRNA.

Northern Blot Analysis

For determination of mRNA levels by Northern blot analysis, total RNAwas prepared from cells by the guanidinium isothiocyanate procedure(Monia et al., Proc. Natl. Acad. Sci. USA, 1996, 93, 15481-15484) 24 hafter initiation of oligonucleotide treatment. Total RNA was isolated bycentrifugation of the cell lysates over a CsCl cushion. Northern blotanalysis, RNA quantitation and normalization to G#PDH mRNA levels weredone according to a reported procedure (Dean and McKay, Proc. Natl.Acad. Sci. USA, 1994, 91, 11762-11766). In bEND cells the2-5-linked-3′-O-methoxyethyl oligonucleotides showed reduction of c-rafmessage activity as a function of concentration. The fact that thesemodified oligonucleotides retained activity promises reduced frequencyof dosing with these oligonucleotides which also show increased in vivonuclease resistance. All 2′,5′-linked oligonucleotides retained theactivity of parent 11061 (Table III) oligonucleotide and improved theactivity even further. A graph of the effect of the oligonucleotides ofthe present invention on c-raf expression (compared to control) in bENDcells is shown in FIG. 7.

EXAMPLE 58

Synthesis of MMI-containing Oligonucleotides

a. Bis-2′-O-methyl MMI Building Blocks

The synthesis of MMI (i.e., R═CH₃) dimer building blocks have beenpreviously described (see, e.g., Swayze, et al., Synlett 1997, 859;Sanghvi, et al., Nucleosides & Nucleotides 1997, 16 907; Swayze, et al.,Nucleosides & Nucleotides 1997, 16, 971; Dimock, et al., Nucleosides &Nucleotides 1997, 16, 1629). Generally,5′-O-(4,4′-dimethoxytrityl)-2′-O-methyl-3′-C-formyl nucleosides werecondensed with 5′-O-(N-methylhydroxylamino)-2′-O-methyl-3′-O-TBDPSnucleosides using 1 equivalent of BH₃ pyridine/1 equivalent ofpyridinium para-toluene sulfonate (PPTS) in 3:1 MeOH/THF. The resultantMMI dimer blocks were then deprotected at the lower part of the sugarwith 15 equivalents of Et₃N-2HF in THF. Thus the T*G^(iBU) dimer unitwas synthesized and phosphitylated to give T*G(MMI)phosphoramidite. In asimilar fashion, A^(BZ)*T(MMI) dimer was synthesized, succinylated andattached to controlled pore glass.

b. Oligonucleotide Synthesis

Oligonucleotides were synthesized on a Perseptive Biosystems Expedite8901 Nucleic Acid Synthesis System. Multiple 1-μmol syntheses wereperformed for each oligonucleotide. A*_(MMI)T solid support was loadedinto the column. Trityl groups were removed with trichloroacetic acid(975 μL over one minute) followed by an acetonitrile wash. Theoligonucleotide was built using a modified thioate protocol. Standardamidites were delivered (210 μL) over a 3 minute period in thisprotocol. The T*_(MMI)G amidite was double coupled using 210 μL over atotal of 20 minutes. The amount of oxidizer,3H-1,2-benzodithiole-3-one-1,1-dioxide (Beaucage reagent, 3.4 g Beaucagereagent/200 mL acetonitrile), was 225 μL (one minute wait step). Theunreacted nucleoside was capped with a 50:50 mixture oftetrahydrofuran/acetic anhydride and tetrahydrofuran/pyridine/1-methylimidazole. Trityl yields were followed by the trityl monitor during theduration of the synthesis. The final DMT group was left intact. Afterthe synthesis, the contents of the synthesis cartridge (1 μmole) weretransferred to a Pyrex vial and the oligonucleotide was cleaved from thecontrolled pore glass (CPG) using 5 mL of 30% ammonium hydroxide (NH₄OH)for approximately 16 hours at 55° C.

c. Oligonucleotide Purification

After the deprotection step, the samples were filtered from CPG usingGelman 0.45 μm nylon acrodisc syringe filters. Excess NH₄OH wasevaporated away in a Savant AS160 automatic SpeedVac. The crude yieldwas measured on a Hewlett Packard 8452A Diode Array Spectrophotometer at260 nm. Crude samples were then analyzed by mass spectrometry (MS) on aHewlett Packard electrospray mass spectrometer. Trityl-onoligonucleotides were purified by reverse phase preparative highperformance liquid chromatography (HPLC). HPLC conditions were asfollows: Waters 600E with 991 detector; Waters Delta Pak C4 column(7.8×300 mm); Solvent A: 50 mM triethylammonium acetate (TEA-Ac), pH7.0; B: 100% acetonitrile; 2.5 mL/min flow rate; Gradient: 5% B forfirst five minutes with linear increase in B to 60% during the next 55minutes. Fractions containing the desired product (retention time=41min. for DMT-ON-16314; retention time 42.5 min. for DMT-ON-16315) werecollected and the solvent was dried off in the SpeedVac.Oligonucleotides were detritylated in 80% acetic acid for approximately60 minutes and lyophilized again. Free trityl and excess salt wereremoved by passing detritylated oligonucleotides through Sephadex G-25(size exclusion chromatography) and collecting appropriate samplesthrough a Pharmacia fraction collector. The solvent was again evaporatedaway in a SpeedVac. Purified oligonucleotides were then analyzed forpurity by CGE, HPLC (flow rate: 1.5 mL/min; Waters Delta Pak C4 column,3.9×300 mm), and MS. The final yield was determined by spectrophotometerat 260 nm.

The synthesized oligonucleotides and their physical characteristics areshown, respectively, in Tables VIII and IX. All nucleosides with anasterisk contain MMI linkage.

TABLE VIII ICAM-1 Oligonucleotides Containing MMI Dimers Synthesized forin Vivo Nuclease and Pharmacology Studies. SEQ ID Back- NO.# (ISIS)#Sequence (5′-3′) bone 2′-Chemistry 21 (16134) TGC ATC CCC CAG GCC ACC P= S, MMI Bis-2′-OMe-MMI, A*T 2′-H 22 (16315) T*GC ATC CCC CAG GCC P = S,MMI Bis-2′-OMe-MMI, ACCA*T2′-H 23 (3082) TGC ATC CCC CAG GCG ACC P = S2′-H, single AT mismatch 23 (13001) TGC ATC CCC CAG GCC ACC P = S 2′-HAT

HPLC conditions were as follows: Waters 600E with detector 991; WatersC4 column (3.9×300 mm); Solvent A: 50 mM TEA-Ac, pH 7.0; B: 100%acetonitrile; 1.5 mL/min flow rate; Gradient: 5% B for first fiveminutes with linear increase in B to 60% during the next 55 minutes.

TABLE IX Physical Characteristics of MMI Oligomers Synthesized forPharmacology, and In Vivo Nuclease Studies SEQ ID Observed HPLC NO.(ISIS) Sequence (5′-3′) Mass Time # # Expected Mass (g) (g) (min) Retn.21 (16314) TGC ATC CCC CAG 6295 6297 23.9 GCC ACC A*T 22 (16315) T*G CATC CCC CAG 6302 6303 24.75 GCC ACC A*T HPLC Conditions: Waters 600Ewith detector 991; Waters C4 column (3.9 × 300 mm); Solvent A: 50 mMTEA-Ac, pH 7.0; B: 100% acetonitrile; 1.5 mL/min. flow rate; Gradient:5% B for first five minutes with linear increase in B to 60% during thenext 55 minutes.

EXAMPLE 59

Synthesis of Sp Terminal Oligonucleotide

a. 3′-O-t-Butyldiphenylsilyl-thymidine (1)

5′-O-Dimethoxytritylthymidine is silylated with 1 equivalent oft-butyldiphenylsilyl chloride (TBDPSC1) and 2 equivalents of imidazolein DMF solvent at room temperature. The 5′-protecting group is removedby treating with 3% dichloracetic acid in CH₂Cl₂.

b. 5′-O-Dimethoxytrityl-thymidin-3′-O-yl-N,N-diisopropylamino(S-pivaloyl-2-mercaptoethoxy)phosphoramidite (2)

5′-O-Dimethoxytrityl thymidine is treated withbis-(N,N-diisopropylamino)-S-pivaloyl-2-mercaptoethoxy phosphoramiditeand tetrazole in CH₂Cl₂/CH₃CN as described by Guzaev et al., Bioorganic& Medicinal Chemistry Letters 1998, 8, 1123) to yield the titlecompound.

c. 5′-O-Dimethoxytrityl-2′-deoxy-adenosin-3′-O-yl-N,N-diisopropylamino(S-pivaloyl-2-mercapto ethoxy)phosphoramidite (3)

5′-O-Dimethoxytrityl-N-6-benzoyl-2′-deoxy-adenosine is phosphitylated asin the previous example to yield the desired amidite.

d. 3′-O-t-Butyldiphenylsilyl-2′-deoxy-N₂-isobutyryl-guanosine (4)

5′-O-Dimethoxytrityl-2′-deoxy-N₂-isobutyryl-guanisine is silylated withTBDPSCl and imidazole in DMF. The 5′-DMT is then removed with 3% DCA inCH₂Cl₂.

e. T_((Sp))G Dimers and T_((S)) Phosphoramidite

Compounds 4 and 2 are condensed (1:1 equivalents) using 1H-tetrazole inCH₃CN solvent followed by sulfuirization employing Beaucage reagent(see, e.g. Iyer, et al., J. Org. Chem. 1990, 55, 4693). The dimers (TG)are separated by column chromatography and the silyl group isdeprotected using t-butyl ammonium fluoride/THF to give Rp and Sp dimersof T_(s)G. Small amounts of these dimers are completely deprotected andtreated with either P1 nuclease or snake venom phosphodiesterase. The Risomer is resistant to P1 nuclease and hydrolyzed by SVPD. The S isomeris resistant to SVPD and hydrolyzed P1 nuclease. The Sp isomer of thefully protected T_(s)G dimer is phosphitylated to giveDMT-T-Sp-G-phosphoramidite.

f. A_(s)T Dimers and Solid Support Containing A_(SP)T Dimer

Compounds 3 and 1 are condensed using 1H-tetrazole in CH₃CN solventfollowed by sulfuirization to give AT dimers. The dimers are separatedby column chromatography and the silyl group is deprotected withTBAF/THF. The configurational assignments are done generally as in theprevious example. The Sp isomer is then attached to controlled poreglass according to standard procedures to give DMT-A_(S)-T-CPGoligomerization with chirally pure Sp dimer units at the termini.

g. Oligonucleotide Synthesis

The oligonucleotide having the sequence T*GC ATC CCC CAG GCC ACC A*T SEQID NO: 22 is synthesized, where T*G and A*T represent chiral Sp dimerblocks described above. DMT-A_(SP)-T-CPG is taken in the synthesiscolumn and the next 16b residues are built using standardphosphorothioate protocols and 3H-1,2-benzodithiol-3-one 1,1 dioxide asthe sulfurizing agent. After building this 18 mer unit followed by finaldetritylation, the chiral Sp dimer phosphoramidite of 5′-DMT-T_(SP)-Gamidite is coupled to give the desired antisense oligonucleotide. Thiscompound is then deprotected in 30% NH₄OH over 16 hours and the oligomerpurified in HPLC and desalted in Sephader G-25 column. The finaloligomer has Sp configuration at the 5′-terminus and 3′-terminus and theinterior has diastereomeric mixture of Rp and Sp configurations.

EXAMPLE 60

Evaluation of in vivo Stability of MMI Capped Oligonucleotides MouseExperiment Procedures

For each oligonucleotide tested, 9 male BALB/c mice (Charles River,Wilmington, Mass.), weighing about 25 g was used (Crooke et al., J.Pharmacol. Exp. Ther., 1996, 277, 923). Following a 1-week acclimation,mice received a single tail vein injection of oligonucleotide (5 mg/kg)administered in phosphate buffered saline (PBS), pH 7.0 One retroorbitalbleed (either 0.25, 0.5, 2 or 4 lv post dose) and a terminal bleed(either 1, 3, 8 or 24 h post dose) were collected from each group. Theterminal bleed (approximately 0.6-0.8 mL) was collected by cardiacpuncture following ketamine/xylazine anesthesia. The blood wastransferred to an EDTA-coated collection tube and centrifuged to obtainplasma. At termination, the liver and kidneys were collected from eachmouse. Plasma and tissues homogenates were used for analysis fordetermination of intact oligonucleotide content by CGE. All samples wereimmediately frozen on dry ice after collection and stored at −80° C.until analysis.

The capillary gel electrophoretic analysis indicated the relativenuclease resistance of MMI capped oligomers compared to ISIS 3082(uniform 2′-deoxy phosphorothioate). Because of the resistance of MMIlinkage to nucleases, the compound 16314 was found to be stable inplasma while 3082 was not. However, in kidney and liver, the compound16314 also showed certain amount of degradation. This implied that while3′-exonuclease is important in plasma, 5′-exonucleases or endonucleasesmay be active in tissues. To distinguish between these twopossibilities, the data from 16315 was analyzed. In plasma as well as intissues, (liver and kidney) the compound was stable in various timepoints. (1, 3 and 24 hrs.). The fact that no degradation was detectedproved that 5′-exonucleases and 3′-exonuclease are prevalent in tissuesand endonucleases are not active. Furthermore, a single linkage (MMI orSp thioate linkage) is sufficient as a gatekeeper against nucleases.

Control of ICAM-1 Expression Cells and Reagents

The bEnd.3 cell line, a brain endothelioma, was the kind gift of Dr.Werner Risau (Max-Planck Institute). Opti-MEM, trypsin-EDTA and DMEMwith high glucose were purchased from Gibco-BRL (Grand Island, N.Y.).Dulbecco's PBS was purchased from Irvine Scientific (Irvine, Calif.).Sterile, 12 well tissue culture plates and Facsflow solution werepurchased from Becton Dickinson (Mansfield, Mass.). Ultrapureformaldehyde was purchased from Polysciences (Warrington, Pa.).Recombinant human TNF-a was purchased from R&D Systems (Minneapolis,Minn.). Mouse interferon-γ was purchased from Genzyme (Cambridge,Mass.). Fraction V, BSA was purchased from Sigma (St. Louis, Mo.). Themouse ICAM-1-PE, VCAM-1-FITC, hamster IgG-FITC and rat IgG_(2a)-PEantibodies were purchased from Pharmingen (San Diego, Calif.).Zeta-Probe nylon blotting membrane was purchased from Bio-Rad (Richmond,Calif.). QuickHyb solution was purchased from Stratagene (La Jolla,Calif.). A cDNA labeling kit, Prime-a-Gene, was purchased from ProMega(Madison, Wis.). NAP-5 columns were purchased from Pharmacia (Uppsala,Sweden).

Oligonucleotide Treatment

Cells were grown to approximately 75% confluency in 12 well plates withDMEM containing 4.5 g/L glucose and 10% FBS. Cells were washed 3 timeswith Opti-MEM pre-warmed to 37° C. Oligonucleotide was premixed withOpti-MEM, serially diluted to desired concentrations and transferredonto washed cells for a 4 hour incubation at 37° C. Media was removedand replaced with normal growth media with or without 5 ng/mL TNF-α and200 U/mL interferon-γ, incubated for 2 hours for northern blot analysisof mRNA or overnight for flow cytometric analysis of cell surfaceprotein expression.

Flow Cytometry

After oligonucleotide treatment, cells were detached from the plateswith a short treatment of trypsin-EDTA (1-2 min.). Cells weretransferred to 12×75 mm polystyrene tubes and washed with 2% BSA, 0.2%sodium azide in D-PBS at 4° C. Cells were centrifuged at 1000 rpm in aBeckman GPR centrifuge and the supernatant was then decanted. ICAM-1,VCAM-1 and the control antibodies were added at 1 ug/mL in 0.3 mL of theabove buffer. Antibodies were incubated with the cells for 30 minutes at4° C. in the dark, under gentle agitation. Cells were washed again asabove and then resuspended in 0.3 mL of FacsFlow buffer with 0.5%ultrapure formaldehyde. Cells were analyzed on a Becton DickinsonFACScan. Results are expressed as percentage of control expression,which was calculated as follows: [((CAM expression foroligonucleotide-treated cytokine induced cells)—(basal CAMexpression))/((cytokine-induced CAM expression)—(basal CAMexpression))]×100. For the experiments involving cationic lipids, bothbasal and cytokine-treated control cells were pretreated with Lipofectinfor 4 hours in the absence of oligonucleotides.

The results reveal the following: 1) Isis 3082 showed an expected doseresponse (25-200 nM); 2) Isis 13001 lost its ability to inhibit ICAM-1expression as expected from a mismatch compound, thus proving anantisense mechanism; 3) 3′-MMI capped oligomer 16314 improved theactivity of 3082, and at 200 nM concentration, nearly twice as active as3082; 4) 5′- and 3′-MMI capped oligomer is the most potent compound andit is nearly 4 to 5 times more efficacious than the parent compound at100 and 200 nM concentrations. Thus, improved nuclease resistanceincreased the potency of the antisense oligonucleotides.

EXAMPLE 61

Control of H-ras Expression

Antisense oligonucleotides targeting the H-ras message were tested fortheir ability to inhibit production of H-ras mRNA in T-24 cells. Forthese test, T-24 cells were plated in 6-well plates and then treatedwith various escalating concentrations of oligonucleotide in thepresence of cationic lipid (Lipofectin, GIBCO) at the ratio of 2.5 μg/mlLipofectin per 100 nM oligonucleotide. Oligonucleotide treatment wascarried out in serum free media for 4 hours. Eighteen hours aftertreatment the total RNA was harvested and analyzed by northern blot forH-ras mRNA and control gene G3PDH. The data is presented in FIGS. 8 and9 in bar graphs as percent control normalized for the G3PDH signal. Ascan be seen, the oligonucleotide having a single MMI linkage in each ofthe flank regions showed significant reduction of H-ras mRNA.

EXAMPLE 62

5-Lipoxygenase Analysis and Assays

A. Therapeutics

For therapeutic use, an animal suspected of having a diseasecharacterized by excessive or abnormal supply of 5-lipoxygenase istreated by administering a compound of the invention. Persons ofordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Such treatment is generallycontinued until either a cure is effected or a diminution in thediseased state is achieved. Long term treatment is likely for somediseases.

B. Research Reagents

The oligonucleotides of the invention will also be useful as researchreagents when used to cleave or otherwise modulate 5-lipoxygenase mRNAin crude cell lysates or in partially purified or wholly purified RNApreparations. This application of the invention is accomplished, forexample, by lysing cells by standard methods, optimally extracting theRNA and then treating it with a composition at concentrations ranging,for instance, from about 100 to about 500 ng per 10 Mg of total RNA in abuffer consisting, for example, of 50 mm phosphate, pH ranging fromabout 4-10 at a temperature from about 30 to about 50° C. The cleaved5-lipoxygenase RNA can be analyzed by agarose gel electrophoresis andhybridization with radiolabeled DNA probes or by other standard methods.

C. Diagnostics

The oligonucleotides of the invention will also be useful in diagnosticapplications, particularly for the determination of the expression ofspecific mRNA species in various tissues or the expression of abnormalor mutant RNA species. In this example, while the macromolecules targeta abnormal mRNA by being designed complementary to the abnormalsequence, they would not hybridize to normal mRNA. Tissue samples can behomogenized, and RNA extracted by standard methods. The crude homogenateor extract can be treated for example to effect cleavage of the targetRNA. The product can then be hybridized to a solid support whichcontains a bound oligonucleotide complementary to a region on the 5′side of the cleavage site. Both the normal and abnormal 5′ region of themRNA would bind to the solid support. The 3′ region of the abnormal RNA,which is cleaved, would not be bound to the support and therefore wouldbe separated from the normal mRNA.

Targeted mRNA species for modulation relates to 5-lipoxygenase; however,persons of ordinary skill in the art will appreciate that the presentinvention is not so limited and it is generally applicable. Theinhibition or modulation of production of the enzyme 5-lipoxygenase isexpected to have significant therapeutic benefits in the treatment ofdisease. In order to assess the effectiveness of the compositions, anassay or series of assays is required.

D. In Vitro Assays

The cellular assays for 5-lipoxygenase preferably use the humanpromyelocytic leukemia cell line HL-60. These cells can be induced todifferentiate into either a monocyte like cell or neutrophil like cellby various known agents. Treatment of the cells with 1.3% dimethylsulfoxide, DMSO, is known to promote differentiation of the cells intoneutrophils. It has now been found that basal HL-60 cells do notsynthesize detectable levels of 5-lipoxygenase protein or secreteleukotrienes (a downstream product of 5-lipoxygenase). Differentiationof the cells with DMSO causes an appearance of 5-lipoxygenase proteinand leukotriene biosynthesis 48 hours after addition of DMSO. Thusinduction of 5-lipoxygenase protein synthesis can be utilized as a testsystem for analysis of oligonucleotides which interfere with5-lipoxygenase synthesis in these cells.

A second test system for oligonucleotides makes use of the fact that5-lipoxygenase is a “suicide” enzyme in that it inactivates itself uponreacting with substrate. Treatment of differentiated HL-60 or othercells expressing 5 lipoxygenase, with 10 μM A23187, a calcium ionophore,promotes translocation of 5-lipoxygenase from the cytosol to themembrane with subsequent activation of the enzyme. Following activationand several rounds of catalysis, the enzyme becomes catalyticallyinactive. Thus, treatment of the cells with calcium ionophoreinactivates endogenous 5-lipoxygenase. It takes the cells approximately24 hours to recover from A23187 treatment as measured by their abilityto synthesize leukotriene B₄. Macromolecules directed against5-lipoxygenase can be tested for activity in two HL-60 model systemsusing the following quantitative assays. The assays are described fromthe most direct measurement of inhibition of 5-lipoxygenase proteinsynthesis in intact cells to more downstream events such as measurementof 5-lipoxygenase activity in intact cells.

A direct effect which oligonucleotides can exert on intact cells andwhich can be easily be quantitated is specific inhibition of5-lipoxygenase protein synthesis. To perform this technique, cells canbe labeled with ³⁵S-methionine (50 μCi/mL) for 2 hours at 37° C. tolabel newly synthesized protein. Cells are extracted to solubilize totalcellular proteins and 5-lipoxygenase is immunoprecipitated with5-lipoxygenase antibody followed by elution from A Sepharose beads. Theimmunoprecipitated proteins are resolved by SDS-protein polyacrylamidegel electrophoresis and exposed for autoradiography. The amount ofimmunoprecipitated 5-lipoxygenase is quantitated by scanningdensitometry.

A predicted result from these experiments would be as follows. Theamount of 5-lipoxygenase protein immunoprecipitated from control cellswould be normalized to 100%. Treatment of the cells with 1 μM, 10 μM,and 30 μM of the macromolecules of the invention for 48 hours wouldreduce immunoprecipitated 5-lipoxygenase by 5%, 25% and 75% of control,respectively.

Measurement of 5-lipoxygenase enzyme activity in cellular homogenatescould also be used to quantitate the amount of enzyme present which iscapable of synthesizing leukotrienes. A radiometric assay has now beendeveloped for quantitating 5-lipoxygenase enzyme activity in cellhomogenates using reverse phase HPLC. Cells are broken by sonication ina buffer containing protease inhibitors and EDTA. The cell homogenate iscentrifuged at 10,000×g for 30 min and the supernatants analyzed for5-lipoxygenase activity. Cytosolic proteins are incubated with 10 μM¹⁴C-arachidonic acid, 2 mM ATP, 50 μM free calcium, 100 μg/mLphosphatidylcholine, and 50 mM bis-Tris buffer, pH 7.0, for 5 min at 37°C. The reactions are quenched by the addition of an equal volume ofacetone and the fatty acids extracted with ethyl acetate. The substrateand reaction products are separated by reverse phase HPLC on a NovapakC18 column (Waters Inc., Millford, Mass.). Radioactive peaks aredetected by a Beckman model 171 radiochromatography detector. The amountof arachidonic acid converted into di-HETE's and mono-HETE's is used asa measure of 5-lipoxygenase activity.

A predicted result for treatment of DMSO differentiated HL-60 cells for72 hours with effective the macromolecules of the invention at 1 μM, 10μM, and 30 μM would be as follows. Control cells oxidize 200 pmolarachidonic acid/5 min/10⁶ cells. Cells treated with 1 μM, 10 μM, and 30μM of an effective oligonucleotide would oxidize 195 pmol, 140 pmol, and60 pmol of arachidonic acid/5 min/10⁶ cells respectively.

A quantitative competitive enzyme linked immunosorbant assay (ELISA) forthe measurement of total 5-lipoxygenase protein in cells has beendeveloped. Human 5-lipoxygenase expressed in E. coli and purified byextraction, Q-Sepharose, hydroxyapatite, and reverse phase HPLC is usedas a standard and as the primary antigen to coat microtiter plates.Purified 5-lipoxygenase (25 ng) is bound to the microtiter platesovernight at 4° C. The wells are blocked for 90 min with 5% goat serumdiluted in 20 mM Tris!HCL buffer, pH 7.4, in the presence of 150 mM NaCl(TBS). Cell extracts (0.2% Triton X-100, 12,000×g for 30 min.) orpurified 5-lipoxygenase were incubated with a 1:4000 dilution of5-lipoxygenase polyclonal antibody in a total volume of 100 μL in themicrotiter wells for 90 min. The antibodies are prepared by immunizingrabbits with purified human recombinant 5-lipoxygenase. The wells arewashed with TBS containing 0.05% tween 20 (TBST), then incubated with100 μL of a 1:1000 dilution of peroxidase conjugated goat anti-rabbitIgG (Cappel Laboratories, Malvern, Pa.) for 60 min at 25° C. The wellsare washed with TBST and the amount of peroxidase labeled secondantibody determined by development with tetramethylbenzidine.

Predicted results from such an assay using a 30 mer oligonucleotide at 1μM, 10 μM, and 30 μM would be 30 ng, 18 ng and 5 ng of 5-lipoxygenaseper 10⁶ cells, respectively with untreated cells containing about 34 ng5-lipoxygenase.

A net effect of inhibition of 5-lipoxygenase biosynthesis is adiminution in the quantities of leukotrienes released from stimulatedcells. DMSO-differentiated HL-60 cells release leukotriene B4 uponstimulation with the calcium ionophore A23187. Leukotriene B4 releasedinto the cell medium can be quantitated by radioimmunoassay usingcommercially available diagnostic kits (New England Nuclear, Boston,Mass.). Leukotriene B4 production can be detected in HL-60 cells 48hours following addition of DMSO to differentiate the cells into aneutrophil-like cell. Cells (2×10⁵ cells/mL) will be treated withincreasing concentrations of the macromolecule for 48-72 hours in thepresence of 1.3% DMSO. The cells are washed and resuspended at aconcentration of 2×10⁶ cell/mL in Dulbecco's phosphate buffered salinecontaining 1% delipidated bovine serum albumin. Cells are stimulatedwith 10 μM calcium ionophore A23187 for 15 min and the quantity of LTB4produced from 5×10⁵ cell determined by radioimmunoassay as described bythe manufacturer.

Using this assay the following results would likely be obtained with anoligonucleotide directed to the 5-LO mRNA. Cells will be treated for 72hours with either 1 μM, 10 μM or 30 μM of the macromolecule in thepresence of 1.3% DMSO. The quantity of LTB₄ produced from 5×10⁵ cellswould be expected to be about 75 pg, 50 pg, and 35 pg, respectively withuntreated differentiated cells producing 75 pg LTB₄.

E. In Vivo Assay

Inhibition of the production of 5-lipoxygenase in the mouse can bedemonstrated in accordance with the following protocol. Topicalapplication of arachidonic acid results in the rapid production ofleukotriene B₄, leukotriene C₄ and prostaglandin E₂ in the skin followedby edema and cellular infiltration. Certain inhibitors of 5-lipoxygenasehave been known to exhibit activity in this assay. For the assay, 2 mgof arachidonic acid is applied to a mouse ear with the contralateral earserving as a control. The polymorphonuclear cell infiltrate is assayedby myeloperoxidase activity in homogenates taken from a biopsy 1 hourfollowing the administration of arachidonic acid. The edematous responseis quantitated by measurement of ear thickness and wet weight of a punchbiopsy. Measurement of leukotriene B₄ produced in biopsy specimens isperformed as a direct measurement of 5-lipoxygenase activity in thetissue. Oligonucleotides will be applied topically to both ears 12 to 24hours prior to administration of arachidonic acid to allow optimalactivity of the compounds. Both ears are pretreated for 24 hours witheither 0.1 μmol, 0.3 μmol, or 1.0 μmol of the macromolecule prior tochallenge with arachidonic acid. Values are expressed as the mean forthree animals per concentration. Inhibition of polymorphonuclear cellinfiltration for 0.1 μmol, 0.3 μmol, and 1 μmol is expected to be about10%, 75% and 92% of control activity, respectively. Inhibition of edemais expected to be about 3%, 58% and 90%, respectively while inhibitionof leukotriene B₄ production would be expected to be about 15%, 79% and99%, respectively.

EXAMPLE 63

5′-O-DMT-2′-deoxy-2′-methylene5-methyluridine-3′-(2-cyanoethyl-N,N-diisoproppyl)phosphoramidite

2′-Deoxy-2′-methylene-3 ′,5′-O-(tetraisopropyldisiloxane-1,3,diyl)-5-methyl uridine is synthesized following theprocedures reported for the corresponding uridine derivative (Hansske,F.; Madej, D.; Robins, M. J. Tetrahedron (1984) 40, 125; Matsuda, A.;Takenusi, K.; Tanaka, S.; Sasaki, T.; Ueda, T. J. Med. Chem. (1991) 34,812; See also Cory, A. H.; Samano, V.; Robins, M. J.; Cory, J. G.2′-Deoxy-2′-methylene derivatives of adenosine, guanosine, tubercidin,cytidine and uridine as inhibitors of L1210 cell growth in culture.Biochem. Pharmacol. (1994), 47(2), 365-71.)

It is treated with IM TBAF in THF to give 2′-deoxy-2′-methylene-5-methyluridine. It is dissolved in pyridine and treated with DMT-Cl and stirredto give the 5′-O-DMT-2′-deoxy-2′-methylene-5-methyl uridine. Thiscompound is treated with 2-cyanoethyl-N,N-diisopropyl phosphoramiditeand diisopropylaminotetrazolide. In a similar manner the correspondingN-6 benzoyl adenosine, N-4-benzoyl cytosine, N-2-isobutyryl guanosinephosphoramidite derivatives are synthesized.

EXAMPLE 63

Synthesis of 3′-O-4′-C-methyleneribonucleoside

5′-O-DMT-3′-O-4′-C-methylene uridine and 5-methyl uridine aresynthesized and phosphitylated according to the procedure of Obika etal. (Obika et al. Bioorg. Med. Chem. Lett. (1999) 9, 515-158). Theamidites are incorporated into oligonucleotides using the protocolsdescribed above.

EXAMPLE 64

Synthesis of 2′-methylene phosphoramidites

5′-O-DMT-2′-(methyl)-3′-O-(2-cyanoethyl-N,N-diisopropylamine)-5-methyluridine-phosphoramidite,5′-O-DMT-2′-(methyl)-N-6-benzoyl adenosine(3′-O-2-cyanoethyl-N,N-diisopropylamino)phosphoramidite,5′-O-DMT-2′-(methyl)-N2-isoburytylguanosine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite and5′-O-DMT-2′-(methyl)-N-4-benzoylcytidine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidites wereobtained by the phosphitylation of the corresponding nucleosides. Thenucleosides were synthesized according to the procedure described byIribarren, Adolfo M.; Cicero, Daniel O.; Neuner, Philippe J. Resistanceto degradation by nucleases of(2′S)-2′-deoxy-2′-C-methyloligonucleotides, novel potential antisenseprobes. Antisense Res. Dev., (1994), 4(2), 95-8; Schmit, Chantal;Bevierre, Marc-Olivier; De Mesmaeker, Alain; Altmann, Karl-Heinz. “Theeffects of 2′- and 3′-alkyl substituents on oligonucleotidehybridization and stability”. Bioorg. Med. Chem. Lett. (1994), 4(16),1969-74.

The phosphitylation is carried out by using the bisamidite procedure.

EXAMPLE 65

Synthesis of 2′-S-methyl phosphoramidites

5′-O-DMT-2′-S-(methyl)-3′-O-(2-cyanoethyl-N,N-diisopropylamine)-5-methyluridine-phosphoramidite, 5′-O-DMT-2′-S(methyl)-N-6-benzoyl adenosine(3′-O-2-cyanoethyl-N,N-diisopropylamino)phosphoramidite,5′-O-DMT-2′-S-(methyl)-N2-isoburytylguanosine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite and5′-O-DMT-2′-S-(methyl)-N-4-benzoylcytidine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidites wereobtained by the phosphitylation of the corresponding nucleosides. Thenucleosides were synthesized according to the procedure described byFraser et al. (Fraser, A.; Wheeler, P.; Cook, P. D.; Sanghvi, Y. S. J.Heterocycl. Chem. (1993) 31, 1277-1287). The phosphitylation is carriedout by using the bisamidite procedure.

EXAMPLE 66

Synthesis of 2′-O-methyl-β-D-arabinofuranosyl Compounds

2′-O-Methyl-β-D-arabinofuranosyl-thymidine containing oligonucleotideswere synthesized following the procedures of Gotfredson et. al.(Gotfredson, C. H. et. al. Tetrahedron Lett. (1994) 35, 6941-6944;Gotfredson, C. H. et. al. Bioorg. Med. Chem. (1996) 4, 1217-1225).5′-O-DMT-2′-ara-(O-methyl)-3′-O-(2-cyanoethyl-N,N-diisopropylamine)-5-methyluridine-phosphoramidite, 5′-O-DMT-2′-ara-(O-methyl)-N-6-benzoyladenosine (3′-O-2-cyanoethyl-N,N-diisopropylamino)phosphoramidite,5′-O-DMT-2′-ara-(O-methyl)-N2-isoburytylguanosine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite and5′-O-DMT-2′-ara-(O-methyl)-N-4-benzoylcytidine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidites areobtained by the phosphitylation of the corresponding nucleosides. Thenucleosides are synthesized according to the procedure described byGotfredson, C. H. et. al. Tetrahedron Lett. (1994) 35, 6941-6944;Gotfredson, C. H. et. al. Bioorg. Med. Chem. (1996) 4, 1217-1225. Thephosphitylation is carried out by using the bisamidite procedure.

EXAMPLE 67

Synthesis of 2′-fluoro-β-D-arabinofuranosyl Compounds

2′-Fluoro-β-D-arabinofuranosyl oligonucleotides are synthesizedfollowing the procedures by Kois, P. et al., Nucleosides Nucleotides 12,1093,1993 and Darnha et al., J. Am. Chem. Soc., 120, 12976, 1998 andreferences sited therin.5′-O-DMT-2′-ara-(fluoro)-3′-O-(2-cyanoethyl-N,N-diisopropylamine)-5-methyluridine-phosphoramidite, 5′-O-DMT-2′-ara-(fluoro)-N-6-benzoyl adenosine(3′-O-2-cyanoethyl-N,N-diisopropylamino)phosphoramidite,5′-O-DMT-2′-ara-(fluoro)-N2-isoburytylguanosine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite and5′-O-DMT-2′-ara-(fluoro)-N-4-benzoylcytidine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidites areobtained by the phosphitylation of the corresponding nucleosides. Thenucleosides are synthesized according to the procedure described byKois, P. et al., Nucleosides Nucleotides 12, 1093, 1993 and Damha etal., J. Am. Chem. Soc., 120, 12976, 1998. The phosphitylation is carriedout by using the bisamidite procedure.

EXAMPLE 68

Synthesis of 2′-hydroxyl-β-D-arabinofuranosyl Compounds

2′-Hydroxyl-β-D-arabinofuranosyl oligonucleotides are synthesizedfollowing the procedures by Resmini and Pfleiderer Helv. Chim. Acta, 76,158,1993; Schmit et al., Bioorg. Med. Chem. Lett. 4, 1969, 1994 Resmini,M.; Pfleiderer, W. Synthesis of arabinonucleic acid (tANA). Bioorg. Med.Chem. Lett. (1994), 16, 1910.; Resmini, Matthias; Pfleiderer, W.Nucleosides. Part LV. Efficient synthesis of arabinoguanosine buildingblocks (Helv. Chim. Acta, (1994), 77, 429-34; and Damha et al., J. Am.Chem. Soc., 1998, 120, 12976, and references sited therin).

5′-O-DMT-2′-ara-(hydroxy)-3′-O-(2-cyanoethyl-N,N-diisopropylamine)-5-methyluridine-phosphoramidite, 5′-O-DMT-2′-ara-(hydroxy)-N-6-benzoyl adenosine(3′-O-2-cyanoethyl-N,N-diisopropylamino)phosphoramidite,5′-O-DMT-2′-ara-(hydroxy)-N2-isoburytylguanosine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite and5′-O-DMT-2′-ara-(hydroxy)-N-4-benzoylcytidine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphorarnidites areobtained by the phosphitylation of the corresponding nucleosides. Thenucleosides are synthesized according to the procedure described byKois, P. et al., Nucleosides Nucleotides 12, 1093, 1993 and Damha etal., J. Am. Chem. Soc., 120, 12976, 1998. The phosphitylation is carriedout by using the bisamidite procedure.

EXAMPLE 69

Synthesis of Difluoromethylene Compounds

5′-O-DMT-2′-deoxy-2′-difluoromethylene-5-methyluridine-3′-(2-cyanoethyl-N,N-diisopropylphosphoramidite),5′-O-DMT-2′-deoxy-2′-difluoromethylene-N-4-benzoyl-cytidine,5′-O-DMT-2′-deoxy-2′-diflyoromethylene-N-6-benzoyl adenosine, and5′-O-DMT-2′-deoxy-2′-deoxy-2′-difluoroethylene-N₂-isobutyryl guanosineare synthesized following the protocols described by Usman et. al. (U.S.Pat. No. 5,639,649, Jun. 17, 1997).

EXAMPLE 70

Synthesis of5′-O-DMT-2′-deoxy-2′-β-(O-acetyl)-21-α-methyl-N6-benzoyl-adenosine-3′-(2-cyanoethyl-N,N-diisopropylphosphoramidite

5′-O-DMT-2′-deoxy-2′-β-(OH)-2′-α-methyl-adenosine is synthesized fromthe compound 5′-3′-protected-2′-keto-adenosine (Rosenthal, Sprinzl andBaker, Tetrahedron Lett. 4233, 1970; see also Nucleic acid relatedcompounds. A convenient procedure for the synthesis of 2′- and3′-ketonucleosides is shown Hansske et al., Dep. Chem., Univ. Alberta,Edmonton, Can., Tetrahedron Lett. (1983), 24(15), 1589-92.) by Grigandaddition of MeMgI in THF solvent, followed by seperation of the isomers.The 2-β-(OH) is protected as acetate. 5′-3′-acetal group is removed,5′-position dimethoxy, tritylated, N-6 position is benzoylated and then3′-position is phosphitylated to give5′-O-DMT-2′-deoxy-2′-β-(O-acetyl)-2′-α-methyl-N6-benzoyl-adenosine-3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite.

EXAMPLE 71

Synthesis of5′-O-DMT-2′-α-ethynyl-N6-benzoyl-adenosine-3′-(2-cyanoethyl-N,N-diisopropylphosphoramidite

5′-O-DMT-2′-deoxy-2′-β-(OH)-2′-α-ethynyl-adenosine is synthesized fromthe compound 5′-3′-protected-2′-keto-adenosine (Rosenthal, Sprinzl andBaker, Tetrahedron Lett. 4233, 1970) by Grigand addition of Ethynyl-MgIin THF solvent, followed by seperation of the isomers. The 2′-β-(OH) isremoved by Robins' deoxygenation procedure (Robins et al., J. Am. Chem.Soc. (1983), 105, 4059-65. 5′-3′-acetal group is removed, 5′-positiondimethoxytritylated, N-6 position is benzoylated and then 3′-position isphosphitylated to give the title compound.

EXAMPLE 72

2′-O-(guaiacolyl)-5-methyluridine

2-Methoxyphenol (6.2 g, 50 mmol) was slowly added to a solution ofborane in tetrahydrofuran (1 M, 10 mL, 10 mmol) with stirring in a 100mL bomb. Hydrogen gas evolved as the solid dissolvedO-2,2′-anhydro-5-methyluridine (1.2 g, 5 mmol), and sodium bicarbonate(2.5 mg) were added and the bomb was sealed, placed in an oil bath andheated to 155° C. for 36 hours. The bomb was cooled to room temperatureand opened. The crude solution was concentrated and the residuepartitioned between water (200 mL) and hexanes (200 mL). The excessphenol was extracted into hexanes. The aqueous layer was extracted withethyl acetate (3×200 mL) and the combined organic layer was washed oncewith water, dried over anhydrous sodium sulfate and concentrated. Theresidue was purified by silica gel flash column chromatography usingmethanol:methylene chloride (1/10, v/v) as the eluent. Fractions werecollected and the target fractions were concentrated to give 490 mg ofpure product as a white solid. Rf=0.545 in CH₂Cl₂/CH₃OH (10:1). MS/ESfor C₁₇H₂0N₂O₇, 364.4; Observed 364.9.

EXAMPLE 73

5′-Dimethoxytrityl-2′-O-(2-methoxyphenyl)-5-methyluridine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite

2′-O-(guaiacolyl)-5-methyl-uridine is treated with 1.2 equivalents ofdimethoxytrityl chloride (DMT-Cl) in pyridine to yield the5′-O-dimethoxy tritylated nucleoside. After evaporation of the pyridineand work up (CH₂Cl₂/saturated NaHCO₃ solution) the compound is purifiedin a silica gel column. The 5′-protected nucleoside is dissolved inanhydrous methylene chloride and under argon atmosphere,N,N-diisopropylaminohydro-tetrazolide (0.5 equivalents) andbis-N,N-diisopropylamino-2-cyanoethyl-phosphoramidite (2 equivalents)are added via syringe over 1 min. The reaction mixture is stirred underargon at room temperature for 16 hours and then applied to a silicacolumn. Elution with hexane:ethylacetate (25:75) gives the titlecompound.

EXAMPLE 74

5′-Dimethoxytrityl-2′-O-(2-methoxyphenyl)-5-methyluridine-3′-O-succinate

The 5′-protected nucleoside from Example 73 is treated with 2equivalents of succinic anhydride and 0.2 equivalents of4-N,N-dimethylaminopyridine in pyridine. After 2 hours the pyridine isevaporated, the residue is dissolved in CH₂Cl₂ and washed three timeswith 100 mL of 10% citric acid solution. The organic layer is dried overanhydrous MgSO₄ to give the desired succinate. The succinate is thenattached to controlled pore glass (CPG) using established procedures(Pon, R. T., Solid phase supports for oligonucleotide synthesis, inProtocols for Oligonucleotides and Analogs, S. Agrawal (Ed.), HumanaPress: Totawa, N.J., 1993, 465-496).

EXAMPLE 75

5′-Dimethoxytrityl-2′-O-(trans-2-methoxycyclohexyl)-5-methyl uridine

2′-3′-O-Dibutylstannyl-5-methyl uridine (Wagner et al., J. Org. Chem.,1974, 39, 24) is alkylated with trans-2-methoxycyclohexyl tosylate at70° C. in DMF. A 1:1 mixture of 2′-O- and3′-O-(trans-2-methoxycyclohexyl)-5-methyluridine is obtained in thisreaction. After evaporation of the DMF solvent, the crude mixture isdissolved in pyridine and treated with dimethoxytritylchloride (DMT-Cl)(1.5 equivalents). The resultant mixture is purified by silica gel flashcolumn chromatography to give the title compound.

EXAMPLE 76

5′-Dimethoxytrityl-2′-O-(trans-2-methoxycyclohexyl)-5-methyluridine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite

5′-Dimethoxytrityl-2′-O-(trans-2-methoxycyclohexyl)-5-methyl uridine isphosphitylated according to the procedure described above to give therequired phosphoramidite.

EXAMPLE 77

5′-Dimethoxytrityl-2′-O-(trans-2-metboxycyclohexyl)-5-methyluridine-3′-O-(succinyl-amino)CPG

5′-Dimethoxytrityl-2′-O-(trans-2-methoxycyclohexyl)-5-methyl uridine issuccinylated and attached to controlled pore glass to give the solidsupport bound nucleoside.

EXAMPLE 78

trans-2-ureido-cyclohexanol

Trans-2-amino-cyclohexanol (Aldrich) is treated with triphosgene inmethylene chloride (1/3 equivalent). To the resulting solution, excessammonium hydroxide is added to give after work up the title compound.

EXAMPLE 79

2′-O-(trans-2-uriedo-cyclohexyl)-5-methyl uridine

Trans-2-uriedo-cyclohexanol (50 mmol) is added to a solution of boranein tetrahydrofuran (1 M, 10 mL, 10 mmol) while stirring in a 10 mL bomb.Hydrogen gas evolves as the reactant dissolves.O2,2′-Anhydro-5-methyluridine (5 mmol) and sodium bicarbonate (2.5 mg)are added to the bomb and sealed. Then it is heated to 140 for 72 hrs.The bomb is cooled to room temperature and opened. The crude materialwas worked up as illustrated above followed by purification by silicagel flash column chromatography to give the title compound.

EXAMPLE 80

5′-O-(Dimethoxytrityl)2′-O-(trans-2-uriedo-cyclohexyl)3′-O-(2-cyanoethyl,N,N,-diisopropyl)uridinephosphoramidite

2′-O-(trans-2-uriedo-cyclohexyl)-5-methyl uridine tritylated at the5′-OH and phosphitylated at the 3′-OH following the proceduresillustrated in example 2 to give the title compound.

EXAMPLE 81

5′-O-dimethoxytrityl-2′-O-(trans-2-uriedo-cyclohexyl)-5-methyl-3′-O-(succinyl)-aminoCPG uridine

5′-O-dimethoxytrityl-2′-O-(trans-2-uriedo-cyclohexyl)-5-methyl uridineis succinylated and attached to CPG as illustrated above.

EXAMPLE 82

2′-O-(trans-2-methoxy-cyclohexyl)adenosine

Trans-2-methoxycyclopentanol, trans-2-methoxycylcohexanol,trans-2-methoxy-cyclopentyl tosylate and trans-2-methoxy-cyclohexyltosylate are prepared according to reported procedures (Roberts, D. D.,Hendrickson, W., J. Org. Chem., 1967, 34, 2415-2417; J. Org. Chem.,1997, 62, 1857-1859). A solution of adenosine (42.74 g, 0.16 mol) in drydimethylformamide (800 mL) at 5° C. is treated with sodium hydride (8.24g, 60% in oil prewashed thrice with hexanes, 0.21 mol). After stirringfor 30 min, trans-2-methoxycyclohexyl tosylate (0.16 mol) is added over20 minutes at 5° C. The reaction is stirred at room temperature for 48hours, then filtered through Celite. The filtrate is concentrated underreduced pressure followed by coevaporation with toluene (2×100 mL) togive the title compound.

EXAMPLE 83

N⁶-Benzoyl-2′-O-(trans-2-methoxycyclohexyl)adenosine

A solution of 2′-O-(trans-2-methoxy-cyclohexyl)adenosine (0.056 mol) inpyridine (100 mL) is evaporated under reduced pressure to dryness. Theresidue is redissolved in pyridine (560 mL) and cooled in an ice waterbath. Trimethylsilyl chloride (36.4 mL, 0.291 mol) is added and thereaction is stirred at 5° C. for 30 minutes. Benzoyl chloride (33.6 mL,0.291 mol) is added and the reaction is allowed to warm to 25° C. for 2hours and then cooled to 5° C. The reaction is diluted with cold water(112 mL) and after stirring for 15 min, concentrated ammonium hydroxide(112 mL). After 30 min, the reaction is concentrated under reducedpressure (below 30° C.) followed by coevaporation with toluene (2×100mL). The residue is dissolved in ethyl acetate-methanol (400 mL, 9:1)and the undesired silyl by-products are removed by filtration. Thefiltrate is concentrated under reduced pressure and purified by silicagel flash column chromatography (800 g, chloroform-methanol 9:1).Selected fractions are combined, concentrated under reduced pressure anddried at 25° C./0.2 mmHg for 2 h to give the title compound.

EXAMPLE 84

N⁶-Benzoyl-5′-O-(dimethoxytrityl)-2′-O-(trans-2-methoxycyclohexyl)adenosine

A solution of N⁶-benzoyl-2′-O-(trans-2-methoxycyclohexyl)adenosine(0.285 mol) in pyridine (100 mL) is evaporated under reduced pressure toan oil. The residue is redissolved in dry pyridine (300 mL) and4,4′-dimethoxytriphenylmethyl chloride (DMT-Cl, 10.9 g, 95%, 0.31 mol)added. The mixture is stirred at 25° C. for 16 h and then poured onto asolution of sodium bicarbonate (20 g) in ice water (500 mL). The productis extracted with ethyl acetate (2×150 mL). The organic layer is washedwith brine (50 mL), dried over sodium sulfate (powdered) and evaporatedunder reduced pressure (below 40° C.). The residue is chromato-graphedon silica gel (400 g, ethyl acetate-hexane 1:1. Selected fractions werecombined, concentrated under reduced pressure and dried at 25° C./0.2mmHg to give the title compound.

EXAMPLE 85

[N⁶-Benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-O-(trans-2-methoxycyclohexyl)adenosine-3′-O-yl]-N,N-diisopropylamino-cyanoethoxyphosphoramidite

Phosphitylation ofN⁶-benzoyl-5′-O-(dimethoxytrityl)-2′-O-(trans-2-methoxycyclohexyl)adenosinewas performed as illustrated above to give the title compound.

EXAMPLE 86

General Procedures for Chimeric C3′-endo and C2′-endo ModifiedOligonucleotide Synthesis

Oligonucleotides are synthesized on a PerSeptive Biosystems Expedite8901 Nucleic Acid Synthesis System. Multiple 1-mmol syntheses areperformed for each oligonucleotide. The 3′-end nucleoside containingsolid support is loaded into the column. Trityl groups are removed withtrichloroacetic acid (975 mL over one minute) followed by anacetonitrile wash. The oligonucleotide is built using a modified diester(P═O) or thioate (P═S) protocol.

Phosphodiester Protocol

All standard amidites (0.1 M) are coupled over a 1.5 minute time frame,delivering 105 μL material. All novel amidites are dissolved in dryacetonitrile (100 mg of amidite/1 mL acetonitrile) to give approximately0.08-0.1 M solutions. The 2′-modified amidites (both ribo and arabinomonomers) are double coupled using 210 μL over a total of 5 minutes.Total coupling time is approximately 5 minutes (210 mL of amiditedelivered). 1-H-tetrazole in acetonitrile is used as the activatingagent. Excess amidite is washed away with acetonitrile. (1S)-(+)-(10-camphorsulfonyl) oxaziridine (CSO, 1.0 g CSO/8.72 mL dryacetonitrile) is used to oxidize (3 minute wait step) deliveringapproximately 375 μL of oxidizer. Standard amidites are delivered (210μL) over a 3-minute period.

Phosphorothioate Protocol

The 2′-modified amidite is double coupled using 210 μL over a total of 5minutes. The amount of oxidizer, 3H-1,2-benzodithiole-3-one-1,1-dioxide(Beaucage reagent, 3.4 g Beaucage reagent/200 mL acetonitrile), is 225μL (one minute wait step). The unreacted nucleoside is capped with a50:50 mixture of tetrahydrofuran/acetic anhydride andtetrahydrofuran/pyridine/1-methyl imidazole. Trityl yields are followedby the trityl monitor during the duration of the synthesis. The finalDMT group is left intact. After the synthesis, the contents of thesynthesis cartridge (1 mmole) is transferred to a Pyrex vial and theoligonucleotide is cleaved from the controlled pore glass (CPG) using30% ammonium hydroxide (NH₄OH, 5 mL) for approximately 16 hours at 55°C.

Oligonucleotide Purification

After the deprotection step, the samples are filtered from CPG usingGelman 0.45 μm nylon acrodisc syringe filters. Excess NH₄OH isevaporated away in a Savant AS160 automatic speed vac. The crude yieldis measured on a Hewlett Packard 8452A Diode Array Spectrophotometer at260 nm. Crude samples are then analyzed by mass spectrometry (MS) on aHewlett Packard electrospray mass spectrometer. Trityl-onoligonucleotides are purified by reverse phase preparative highperformance liquid chromatography (HPLC). HPLC conditions are asfollows: Waters 600E with 991 detector; Waters Delta Pak C4 column(7.8×300 mm); Solvent A: 50 mM triethylammonium acetate (TEA-Ac), pH7.0; Solvent B: 100% acetonitrile; 2.5 mL/min flow rate; Gradient: 5% Bfor first five minutes with linear increase in B to 60% during the next55 minutes. Fractions containing the desired product/s (retentiontime=41 minutes for DMT-ON-16314; retention time=42.5 minutes forDMT-ON-16315) are collected and the solvent is dried off in the speedvac. Oligonucleotides are detritylated in 80% acetic acid forapproximately 60 minutes and lyophilized again. Free trityl and excesssalt are removed by passing detritylated oligonucleotides throughSephadex G-25 (size exclusion chromatography) and collecting appropriatesamples through a Pharmacia fraction collector. The solvent is againevaporated away in a speed vac. Purified oligonucleotides are thenanalyzed for purity by CGE, HPLC (flow rate: 1.5 mL/min; Waters DeltaPak C4 column, 3.9×300 mm), and MS. The final yield is determined byspectrophotometer at 260 nm.

Procedures

Procedure 1

ICAM-1 Expression

Oligonucleotide Treatment of HUVECs

Cells were washed three times with Opti-MEM (Life Technologies, Inc.)prewarmed to 37° C. Oligonucleotides were premixed with 10 g/mLLipofectin (Life Technologies, Inc.) in Opti-MEM, serially diluted tothe desired concentrations, and applied to washed cells. Basal anduntreated (no oligonucleotide) control cells were also treated withLipofectin. Cells were incubated for 4 h at 37° C., at which time themedium was removed and replaced with standard growth medium with orwithout 5 mg/mL TNF-α 7 & D Systems). Incubation at 37° C. was continueduntil the indicated times.

Quantitation of ICAM-1 Protein Expression by Fluorescence-activated CellSorter

Cells were removed from plate surfaces by brief trypsinization with0.25% trypsin in PBS. Trypsin activity was quenched with a solution of2% bovine serum albumin and 0.2% sodium azide in PBS (+Mg/Ca). Cellswere pelleted by centrifugation (1000 rpm, Beckman GPR centrifuge),resuspended in PBS, and stained with 3 1/10⁵ cells of the ICAM-1specific antibody, CD54-PE (Pharmingin). Antibodies were incubated withthe cells for 30 min at 4C in the dark, under gently agitation. Cellswere washed by centrifugation procedures and then resuspended in 0.3 mLof FacsFlow buffer (Becton Dickinson) with 0.5% formaldehyde(Polysciences). Expression of cell surface ICAM-1 was then determined byflow cytometry using a Becton Dickinson FACScan. Percentage of thecontrol ICAM-1 expression was calculated as follows:[(oligonucleotide-treated ICAM-1 value)-(basal ICAM-1value)/(non-treated ICAM-1 value)-(basal ICAM-1 value)]. (Baker, Brenda,et. al. 2′-O-(2-Methoxy)ethyl-modified Anti-intercellular AdhesionMolecule 1 (ICAM-1) Oligonucleotides Selectively Increase the ICAM-1mRNA Level and Inhibit Formation of the ICAM-1 Translation InitiationComplex in Human Umbilical Vein Endothelial Cells, The Journal ofBiological Chemistry, 272, 11994-12000, 1997.)

ICAM-1 expression of chimeric C3′-endo and C2′-endo modifiedoligonucleotides of the invention is measured by the reduction of ICAM-1levels in treated HUVEC cells. The oligonucleotides are believed to workby RNase H cleavage mechanism. Appropriate scrambled controloligonucleotides are used as controls. They have the same basecomposition as the test sequence.

Sequences that contain the chimeric C3′-endo (2′-MOE) and C2′-endo (oneof the following modifications: 2′-S—Me, 2′-Me, 2′-ara-F, 2′-ara-OH,2′-ara-O—Me) as listed in Table X below are prepared and tested in theabove assay. SEQ ID NO: 24, a C-raf targeted oligonucleotide, is used asa control.

TABLE X Oligonucleotides Containing chimeric 2′-O-(2-methoxyethyl) and2′-S-(methyl) modifica- tions. SEQ ID NO: Sequence (5′-3′) Target 24AsTsGs C ^(m) sAsTs TsCs^(m)Ts GsCs_(m) mouse Cs^(m) Cs^(m)C^(m)sC ^(m)s AsAsGs GsA C-raf 25 GsC^(m)sC^(m)s C^(m)sAsAs GsC^(m)sTs humanGsGsC^(m)s ASTsC^(m)S C^(m)sGSTs ICAM-1 C^(m)SA

All nucleosides in bold are 2′-O-(methoxyethyl); subscript s indicates aphosphorothioate linkage; underlined nucleosides indicate2′-S—Me-modification. Superscript m on C (Cm)indicates a 5-methyl-C.

TABLE XI Oligonucleotides Containing chimeric 2′-O-(2-methoxyethyl) and2′-O-(methyl) modifica- tions SEQ ID NO: Sequence (5′-3′) Target 24AsTsGs C^(m)sAsTs TsCs^(m)Ts mouse GsCs^(m)Cs^(m) Cs^(m)C^(m)sC^(m)sAsAsGs C-raf GsA 25 GsC^(m)sC^(m)s C^(m)sAsAs GsC^(m)sTs humanGsGsC^(m)s ASTsC^(m)S C^(m)sGSTs ICAM-1 C^(m)SA

All nucleosides in bold are 2′-O-(methoxyethyl); subscript s indicates aphosphorothioate linkage; underlined nucleosides indicate 2′-Methylmodification. Superscript m on C (Cm)indicates a 5-methyl-C.

TABLE XII Oligonucleotides Containing chimeric 2′-O-(2-methoxyethyl) and2′-ara-(fluoro) modifi- cations SEQ ID NO: Sequence (5′-3′) Target 24AsTsGs C^(m)sAsTs TsCs^(m)Ts mouse GsCs^(m)Cs^(m) Cs^(m)C^(m)sC^(m)sAsAsGs C-raf GsA 25 GsC^(m)sC^(m)s C^(m)sAsAs GsC^(m)sTs humanGsGsC^(m)s ASTsC^(m)S C^(m)sGSTs ICAM-1 C^(m)SA --

All nucleosides in bold are 2′-O-(methoxyethyl); subscript s indicates aphosphorothioate linkage; underlined nucleosides indicate2′-ara-(fluoro) modification. superscript m on C (Cm)indicates a5-methyl-C.

TABLE XIII Oligonucleotides Containing chimeric 2′-O-(2-methoxyethyl)and 2′-ara-(OH) modifica- tions SEQ ID NO: Sequence (5′-3′) Target 24AsTsGs C^(m)sAsTs TsCs^(m)Ts mouse GsCs^(m)Cs^(m)Cs^(m)C^(m)sC^(m)sAsAsGs C-raf GsA 25 GsC^(m)sC^(m)s C^(m)sAsAs GsC^(m)sTs humanGsGsC^(m)s ASTsC^(m)S C^(m)sGSTs ICAM-1 C^(m)SA

All nucleosides in bold are 2=-O-(methoxyethyl); subscript s indicates aphosphorothioate linkage; underlined nucleosides indicate 2′-ara-(OH)modification. superscript m on C (Cm)indicates a 5-methyl-C.

TABLE XIV Oligonucleotides Containing chimeric 2′-O-(2-methoxyethyl) and2′-ara-(OMe) modifica- tions SEQ ID NO: Sequence (5′-3′) Target 24AsTsGs C^(m)sAsTs TsCs^(m)Ts mouse GsCs^(m)Cs^(m) Cs^(m)C^(m)sC^(m)sAsAsGs C-raf GsA 25 GsC^(m)sC^(m)s C^(m)sAsAs GsC^(m)sTs humanGsGsC^(m)s ASTsC^(m)S C^(m)sGSTs ICAM-1 C^(m)SA-3′

All nucleosides in bold are 2=-O-(methoxyethyl); subscript S indicates aphosphorothioate linkage; underlined nucleosides indicate 2′-ara-(OMe)modification. superscript m on C (C^(m))indicates a 5-methyl-C.

Procedure 2

Enzymatic Degradation of 2′-O-modified Oligonucleotides

Three oligonucleotides are synthesized incorporating the modificationsshown in Table 2 below at the 3′-end. These modified oligonucleotidesare subjected to snake venom phosphodiesterase action.

Oligonucleotides (30 nanomoles) are dissolved in 20 mL of buffercontaining 50 mM Tris-HCl pH 8.5, 14 mM MgCl₂, and 72 mM NaCl. To thissolution 0.1 units of snake-venom phosphodiesterase (Pharmacia,Piscataway, N.J.), 23 units of nuclease P1 (Gibco LBRL, Gaithersberg,Md.), and 24 units of calf intestinal phosphatase (Boehringer Mannheim,Indianapolis, Ind.) are added and the reaction mixture is incubated at37 C. for 100 hours. HPLC analysis is carried out using a Waters model715 automatic injector, model 600E pump, model 991 detector, and anAlltech (Alltech Associates, Inc., Deerfield, Ill.)nucleoside/nucleotide column (4.6×250 mm). All analyses are performed atroom temperature. The solvents used are A: water and B: acetonitrile.Analysis of the nucleoside composition is accomplished with thefollowing gradient: 0-5 min., 2% B (isocratic); 5-20 min., 2% B to 10% B(linear); 20-40 min., 10% B to 50% B. The integrated area per nanomoleis determined using nucleoside standards. Relative nucleoside ratios arecalculated by converting integrated areas to molar values and comparingall values to thymidine, which is set at its expected value for eacholigomer.

TABLE XV Relative Nuclease Resistance of 2′-Modified ChimericOligonucleotides 5′-TTT TTT TTT TTT TTT T*T*T*T*-3′ SEQ ID NO:26(Uniform phosphodiester) T* = 2′-modified T -S-Me -Me -2′-ara-(F)-2′-ara-(OH) -2′-ara-(OMe)Procedure 3General Procedure for the Evaluation of Chimeric C3′-endo and C2′-endoModified Oligonucleotides Targeted to Ha-ras

Different types of human tumors, including sarcomas, neuroblastomas,leukemias and lymphomas, contain active oncogenes of the ras genefamily. Ha-ras is a family of small molecular weight GTPases whosefunction is to regulate cellular proliferation and differentiation bytransmitting signals resulting in constitutive activation of ras areassociated with a high percentage of diverse human cancers. Thus, rasrepresents an attractive target for anticancer therapeutic strategies.

SEQ ID NO: 27 (5′-TsCsCs GsTsCs AsTsCs GsCsTs CsCsTs CsAsGs GsG-3′) is a20-base phosphorothioate oligodeoxynucleotide targeting the initiationof translation region of human Ha-ras and it is a potentisotype-specific inhibitor of Ha-ras in cell culture based on screeningassays (IC₅₀=45 nm). Treatment of cells in vitro with SEQ ID NO: 27results in a rapid reduction of Ha-ras mRNA and protein synthesis andinhibition of proliferation of cells containing an activating Ha-rasmutation. When administered at doses of 25 mg/kg or lower by dailyintraperitoneal injection (IP), SEQ ID NO: 27 exhibits potent antitumoractivity in a variety of tumor xenograft models, whereas mismatchcontrols do not display antitumor activity. SEQ ID NO: 27 has been shownto be active against a variety of tumor types, including lung, breast,bladder, and pancreas in mouse xenograft studies (Cowsert, L. M.Anti-cancer drug design, 1997, 12, 359-371). A second-generation analogof SEQ ID NO: 27, where the 5′ and 3′ termini (“wings”) of the sequenceare modified with 2′-methoxyethyl (MOE) modification and the backbone iskept as phosphorothioate (Table XVI, SEQ ID NO: 27 (5′-TsCsCs GsTsCsAsTsCs GsCsTs CsCsTs CsAsGs GsG-3′)), exhibits IC₅₀ of 15 nm in cellculture assays. thus, a 3-fold improvement in efficacy is observed fromthis chimeric analog. Because of the improved nuclease resistance of the2′-MOE phosphorothioate, SEQ ID NO: 27 (5′-TsCsCs GsTsCs AsTsCs GsCsTsCsCsTs CsAsGs GsG-3′) increases the duration of antisense effect invitro. This will relate to frequency of administration of this drug tocancer patients. SEQ ID NO: 27 (5′-TsCsCs GsTsCs AsTsCs GsCsTs CsCsTsCsAsGs GsG-3′) is currently under evaluation in ras dependent tumormodels (Cowsert, L. M. Anti-cancer drug design, 1997, 12, 359-371). Theparent compound, SEQ ID NO: 27, is in Phase I clinical trials againstsolid tumors by systemic infusion.

Antisense oligonucleotides having the 2′-Me modification are preparedand tested in the aforementioned assays in the manner described todetermine activity.

Ha-ras Antisense Oligonucleotides with chimeric C3′-endo and C2′-endomodifications and Their Controls.

TABLE XVI Ha-ras Antisense Oligonucleotides With chimeric C3′-endo andC2′-endo modifications and Their Con- trols. SEQ ID Back- NO: Sequencebone 2′-Modif. Comments 27 5′-TsCsCs GsTsCs P = S 2′-H parent AsTsCsGsCsTs CsCsTs CsAsGs GsG-3′ 28 5′-TsCsAs GsTsAs P = S 2′-H mismatchAsTsAs GsGsCs CsCsAs CsAsTs GsG-3′ 29 5′-ToToCo GsTsCs AsTs P = O/2′-O-Moe Parent Cs GsCsTs CoCoTo CoAo P = S/ in wings Gapmer Go GoG-3′ P= O (Mixed Backbone) 27 5′-TsCsCs GsTsCs AsTs P = S 2′-O-MOE ParentCs GsCsTs CsCsTs CsAs in wings Gapmer as Gs GsG-3′ uniform thioate 295′-ToCoAo GsTsAs AsTs P = O in wings Gapmer As GsCsCs GsCsCs GsCo (mixedCo CoCoAo CoAoTo GoG- Backbone) 3′ 28 5′-TsCsAs GsTsAs AsTs P = S2′-O-MOE Control As GsCsCs GsCsCs in wings Gapmer as CsCsAs CsAsTsGsC-3′ uniform Thioate 27 5′-TsCsCs GsTsCs AsTs P = S 2′-O-MOE ControlCs GsCsTs CsCsTs CsAs in wings Gapmer Gs GsG-3′ with MOE control 285′-TsCsAs GsTsAs AsTs P = S 2′-O-MOE Control As GsCsCs GsCsCs CsCs inwings Gapmer As CsAsTs GsC-3′ with MOE Control All underlined portionsof sequences are 2′-Me.Procedure 7In vivo Nuclease Resistance

The in vivo Nuclease Resistance of chimeric C3′-endo and C2′-endomodified oligonucleotides is studied in mouse plasma and tissues (kidneyand liver). For this purpose, the C-raf oligonucleotide series SEQ IDNO: 30 are used and the following five oligonucleotides listed in theTable below will be evaluated for their relative nuclease resistance.

TABLE XVII Study of in vivo Nuclease Resistance of chimeric C3′-endo(2′-O-MOE) and C2′-endo (2′-S-Me) modi- fied oligonucleotides with andwithout nuclease resistant caps (2′-5′-phosphate or phosphorothio- atelinkage with 3′-O-MOE in cap ends). SEQ ID Back- NO: Sequence boneDescription 30 5′-ATG CAT TCT GCC P = S, (control) CCA AGGA-3′ 2′-Hrodent C-raf antisense oligo 31 AoToGo CoAsTs TsCsTs P = O/2′-MOE/2′-S-Me/ GsCsCs CsCsAo AoGoGo P = S/ 2′-MOE A P = O 32 AsTsGsCsAsTs TsCsTs P = S 2′-MOE/2′-S-Me/ GsCsCs CsCsAs AsGsGs 2′-MOE A 33Ao*ToGo CoAsTs TsCsTs P = O/ In asterisk, 2′-5′ GsCsCs CsCsAo AoGoGo P= S/ linkage with 3′-O- *A P = O MOE; 2′-O-MOE/2′-S- Me/2′-O-MOE/2′-5′linkage with 3′-O- MOE in asterisk; 34 As*TsGs CsAsTs TsCsTs P = S Inasterisk, 2′-5′ GsCsCs CsCsAs AsGsGs linkage with *A 3′-O-MOE; 2′-O-MOE/2′-S-Me/2′-O- MOE/2′-5′ linkage with 3′-O-MOE in asterisk.

TABLE XVIII Study of in vivo Nuclease Resistance of chimeric C3′-endo(2′-O-MOE) and C2′-endo (2′-Me) modified oligonucleotides with andwithout nuclease resi- stant caps (2′-5′-phosphate or phosphorothioatelinkage with 3′-O-MOE in cap ends). SEQ ID Back- NO: Sequence boneDescription 30 5′-ATG CAT TCT GCC CCA P = S, (control) AGGA-3′ 2′-Hrodent C-raf antisense oligo 31 AoToGo CoAsTs TsCsTs Gs P = O/2′-MOE/2′-Me/ CsCs CsCsAo AoGoGo A′ P = S/ 2′-MOE P = O 32 AsTsGs CsAsTsTsCsTs Gs P = S 2′-MOE/2′-Me/ CsCs CsCsAs AsGsGs A 2′-MOE   33 Ao*ToGoCoAsTs TsCsTs Gs P = O/ In asterisk, 2′- CsCs CsCsAo AoGoGo *A P = S5′ linkage with /P = O 3′-O-MOE; 2′-O- MOE/ 2′-Me/2′-O- MOE/2′-5′ link-age with 3′-O- MOE in asterisk;   34 As*TsGs CsAsTs TsCsTs Gs P = S Inasterisk, 2′- CsCs CsCsAs AsGsGs *A 5′ linkage with 3′-O-MOE; 2′-O- MOE/2′-Me/2′-O- MOE/2′-5′ link- age with 3′-O- MOE in asterisk;

TABLE XIX Study of in vivo Nuclease Resistance of chimeric C3′-endo(2′-O-MOE) and C2′-endo (2′-ara-F) modi- fied oligonucleotides with andwithout nuclease resistant caps (2′-5′-phosphate or phosphorothio- atelinkage with 3′-O-MOE in cap ends). SEQ ID Back- NO: Sequence boneDescription 30 5′-ATG CAT TCT GCC CCA P = S, (control) AGGA-3′ 2′-Hrodent C-raf antisense oligo 31 AoToGo CoAsTs TsCsTs P = O/2′-MOE/2′-ara-F/ GsCsCs CsCsAo AoGoGo A P = S/ 2′-MOE P = O 32 AsTsGsCsAsTs TsCsTs P = S 2′-MOE/2′-ara- CsCsAs GsCsCs AsGsGs A F/2′-MOE 33Ao*ToGo CoAsTs TsCsTs P = O/ In asterisk, 2′- GsCsCs CsCsAo AoGoGo *A P= S/ 5′ linkage with P = O 3′-O-MOE; 2′-O-MOE/ 2′-ara-F/2′-O-MOE/2′-5′ link- age with 3′-O- MOE in asterisk; 34 As*TsGs CsAsTs TsCsTsP = S In asterisk, 2′- GsCsCs CsCsAs AsGsGs *A 5′ linkage with 3′-O-MOE;2′-O -MO 2′-ara-F/2′-O- MOE/2′-5′ link- age with 3′-O- MOE in asterisk;

TABLE XX Study of in vivo Nuclease Resistance of chimeric C3′-endo(2′-O-MOE) and C2′-endo (2′-ara-OH) modi- fied oligonucleotides with andwithout nuclease resistant caps (2′-5′-phosphate or phosphorothio- atelinkage with 3′-O-MOE in cap ends). SEQ ID Back- NO: Sequence boneDescription 30 5′-ATG CAT TCT GCC CCA P = S, (control) AGGA-3′ 2′-Hrodent C-raf antisense oligo 31 AoToGo CoAsTs TsCsTs P = O/2′-MOE/2′-ara- GsCsCs CsCsAo AoGoGo A P = S/ OH/2′-MOE P = O 32 AsTsGsCsAsTs TsCsTs P = S 2′-MOE/2′-ara- GsCsCs CsCsAs AsGsGs A OH/2′-MOE 33Ao*ToGo CoAsTs TsCsTs P = O/ In asterisk, 2′- GsCsCs CsCsAo AoGoGo *A P= S/ 5′ linkage with P = O 3′-O-MOE; 2′-O- MOE/2′-ara-OH/ 2′-O-MOE/2′-5′ link- age with 3′-O- MOE in asterisk; 34 As*TsGs CsAsTs TsCsTsP = S In asterisk, 2′- GsCsCs CsCsAs AsGsGs *A 5′ linkage with 3′-O-MOE;2′-O- MOE/2′-ara-OH/ 2′-O-MOE/2′-5′ linkage with 3′- O-MOE in asterisk;

TABLE XXI Study of in vivo Nuclease Resistance of chimeric C 3′-endo(2′-O-MOE) and C2′-endo (2′-ara-OMe) modi- fied oligonucleotides withand without nuclease resistant caps (2′5′-phosphate or phosphorothioatelinkage with 3′-O-MOE in cap ends). SEQ ID Back- NO: Sequence boneDescription 30 5′-ATG CAT TCT GCC CCA P = S, (control) ro- AGG A-3′ 2′-Hdent C-raf antisense oli- 31 AoToGo CoAsTs TsCsTs GsCsCs P = O/ go2′-MOE/2′- CsCsAo AoGoGo Aa P = S/ ara-OMe/2′-MOE P = O 32 AsTsGs CsAsTsTsCsTs GsCsCs P = S 2′-MOE/2′-ara- CsCsAs AsGsGs A OMe/2′-MOE 33 Ao*ToGoCoAsTs TsCsTs GsCsCs P = O/ In asterisk, CsCsAo AoGoGo *A P = S/2′-5′ linkage P = O with 3′-O-MOE; 2′-O-MOE/2′- ara-OMe/2′-O-MOE/2′-5′ link- age with 3′-O- MOE in aste- risk; 34 As*TsGs CsAsTsTsCsTs GsCsCs P = S In asterisk, CsCsAs AsGsGs *A 2′-5′ linkage with3′-O-MOE; 2′-O-MOE/2′- ara-OMe/2′-O- MOE/2′-5′ linkage with 3′-O-MOE inasterisk.Procedure 8Animal Studies for in vivo Nuclease Resistance

For each oligonucleotide to be studied, 9 male BALB/c mice (CharlesRiver, Wilmington, Mass.), weighing about 25 g are used (Crooke et al.,J. Pharmacol. Exp. Ther., 1996, 277, 923). Following a 1-weekacclimation, the mice receive a single tail vein injection ofoligonucleotide (5 mg/kg) administered in phosphate buffered saline(PBS), pH 7.0. The final concentration of oligonucleotide in the dosingsolution is (5 mg/kg) for the PBS formulations. One retro-orbital bleed(either 0.25, 9.05, 2 or 4 post dose) and a terminal bleed (either 1, 3,8 or 24 h post dose) is collected from each group. The terminal bleed(approximately 0.6-0.8 mL) is collected by cardiac puncture followingketamine/xylazine anesthesia. The blood is transferred to an EDTA-coatedcollection tube and centrifuged to obtain plasma. At termination, theliver and kidneys will be collected from each mouse. Plasma and tissueshomogenates will be used for analysis for determination of intactoligonucleotide content by CGE. All samples are immediately frozen ondry ice after collection and stored at −80° C. until analysis.

Procedure 9

RNase H Studies with Chimeric C3′-endo and C2′-endo ModifiedOligonucleotides with and Without Nuclease Resistant Caps

³²P Labeling of Oligonucleotides

The oligoribonucleotide (sense strand) was 5′-end labeled with ³²P using[³²P]ATP, T4 polynucleotide kinase, and standard procedures (Ausubel, F.M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J.A., and Struhl, K., in Current Protocols in Molecular Biology, JohnWiley, New York (1989)). The labeled RNA was purified by electrophoresison 12% denaturing PAGE (Sambrook, J., Frisch, E. F., and Maniatis, T.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Plainview (1989)). The specific activity of the labeledoligonucleotide was approximately 6000 cpm/fmol.

Determination of RNase H Cleavage Patterns

Hybridization reactions were prepared in 120 μL of reaction buffer [20mM Tris-HC (pH 7.5), 20 mM KCl, 10 mM MgCl₂, 0.1 mM DTT] containing 750nM antisense oligonucleotide, 500 nM sense oligoribonucleotide, and100,000 cpm ³²P-labeled sense oligoribonucleotide. Reactions were heatedat 90° C. for 5 min and 1 unit of Inhibit-ACE was added. Samples wereincubated overnight at 37° C. degrees. Hybridization reactions wereincubated at 37° C. with 1.5×10.8⁻⁸ mg of E. coli RNase H enzyme forinitial rate determinations and then quenched at specific time points.Samples were analyzed by trichloroacetic acid (TCA) assay or bydenaturing polyacrylamide gel electrophoresis as previously described[Crooke, S. T., Lemonidis, K. M., Neilson, L., Griffey, R., Lesnik, E.A., and Monia, B. P., Kinetic characteristics of Escherichia coli RNaseH1: cleavage of various antisense oligonucleotide-RNA duplexes, BiochemJ, 312, 599 (1995); Lima, W. F. and Crooke, S. T., Biochemistry 36,390-398, 1997].

Those skilled in the art will appreciate that numerous changes andmodifications can be made to the preferred embodiments of the inventionand that such changes and modifications can be made without departingfrom the spirit of the invention. It is therefore intended that theappended claims cover all such equivalent variations as fall within thetrue spirit and scope of the invention.

1. An oligonucleotide comprising a plurality of nucleotides, wherein: afirst portion of said plurality of nucleotides have B-formconformational geometry and are joined together in a continuoussequence, at least two of said nucleotides of said first portion beingribonucleotides; and a further portion of said plurality of nucleotidesare ribonucleotide that have A-form conformation geometry and are joinedtogether in at least one continuous sequence.
 2. The oligonucleotide ofclaim 1 wherein each nucleotide of said first portion, independently, isa 2′-SCH₃ ribonucleotide, a 2′-NH₂ ribonucleotide, a 2′-NH(C₁-C₂alkyl)ribonucleotide, a 2′-N(C₁-C₂ alkyl)₂ ribonucleotide, a 2′-CF₃ribonucleotide, a 2′=CH₂ ribonucleotide, a 2′=CHF ribonucleotide, a2′=CF₂ ribonucleotide, a 2′-CH₃ ribonucleotide, a 2′-C₂H₅ribonucleotide, a 2′-CH═CH₂ ribonucleotide or a 2′-C≡CH ribonucleotide.3. The oligonucleotide of claim 1 wherein each of said nucleotides ofsaid first portion are joined together in said continuous sequence byphosphate, phosphorothioate, phosphorodithioate or boranophosphatelinkages.
 4. The oligonucleotide of claim 1 wherein each nucleotide ofsaid further portion, independently, is a 2′-fluoro nucleotide or anucleotide having a 2′-substituent having the formula I or II:

wherein E is C₁-C₁₀ alkyl, N(Q₁)(Q₂) or N═C(Q₁)(Q₂); each Q₁ and Q₂ is,independently, H, C₁-C₁₀ alkyl, dialkylaminoalkyl, a nitrogen protectinggroup, a tethered or untethered conjugate group, a linker to a solidsupport, or Q₁ and Q₂, together, are joined in a nitrogen protectinggroup or a ring structure that can include at least one additionalheteroatom selected from N and O; R₃ is OX, SX, or N(X)₂; each X is,independently, H, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C(═NH)N(H)Z, C(═O)N(H)Zor OC(═O)N(H)Z; Z is H or C₁-C₈ alkyl; L₁, L₂ and L₃ form a ring systemhaving from about 4 to about 7 carbon atoms or having from about 3 toabout 6 carbon atoms and 1 or 2 heteroatoms selected from oxygen,nitrogen and sulfur and wherein said ring system is aliphatic,unsaturated aliphatic, aromatic, or saturated or unsaturatedheterocyclic; Y is alkyl or haloalkyl having 1 to about 10 carbon atoms,alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 to about 10carbon atoms, aryl having 6 to about 14 carbon atoms, N(Q₁)(Q₂), O(Q₁),halo, S(Q₁), or CN; each q₁ is, independently, from 2 to 10; each q₂ is,independently, 0 or 1; m is 0, 1 or 2; p is from 1 to 10; and q₃ is from1 to 10 with the proviso that when p is 0, q₃ is greater than
 1. 5. Theoligonucleotide of claim 1 wherein each of said nucleotides of saidfurther portion, independently, is a 2′-F ribonucleotide, a 2′-O—(C₁-C₆alkyl)ribonucleotide, or a 2′-O—(C₁-C₆ substituted alkyl)ribonucleotidewherein the substitution is C₁-C₆ ether, C₁-C₆ thioether, amino,amino(C₁-C₆ alkyl) or amino(C₁-C₆ alkyl)₂.
 6. The oligonucleotide ofclaim 1 wherein all of said nucleotides of said further portion arejoined together in a continuous sequence by 3′-5′ phosphodiester, 2′-5′phosphodiester, phosphorothioate, Sp phosphorothioate, Rpphosphorothioate, phosphorodithioate, 3′-deoxy-3′-aminophosphoroamidate, 3′-methylenephosphonate, methylene(methylimino),dimethylhydrazino, amide 3, amide 4 or boranophosphate linkages.
 7. Theoligonucleotide of claim 1 wherein at least two of said nucleotides ofsaid further portion are joined together in a continuous sequence thatis positioned 3′ to said continuous sequence of said first portion ofsaid plurality of nucleotides.
 8. The oligonucleotide of claim 1 whereinat least two of said nucleotides of said further portion are joinedtogether in a continuous sequence that is positioned 5′ to saidcontinuous sequence of said first portion.
 9. The oligonucleotide ofclaim 1 wherein at least two of said nucleotides of said further portionare joined together in a continuous sequence that is positioned 3′ tosaid continuous sequence of said first portion and at least two of saidfurther portion are joined together in a continuous sequence that ispositioned 5′ to said continuous sequence of said first portion.
 10. Theoligonucleotide of claim 1 wherein each nucleotide of said firstportion, independently, is a 2′-SCH₃ ribonucleotide, a 2′-NH₂ribonucleotide, a 2′-NH(C₁-C₂ alkyl)ribonucleotide, a 2′-N(C₁-C₂ alkyl)₂ribonucleotide, a 2′=CH₂ ribonucleotide, a 2′-CH₃ ribonucleotide, a2′-C₂H₅ ribonucleotide, a 2′-CH═CH₂ ribonucleotide or a 2′-C≡CHribonucleotide.
 11. The oligonucleotide of claim 1 wherein eachnucleotide of said first portion, independently, is a 2′-SCH₃ribonucleotide, a 2′-NH₂ ribonucleotide a 2′-NH(C₁-C₂alkyl)ribonucleotide, a 2′-N(C₁-C₂ alkyl)₂ ribonucleotide or a 2′-CH₃ribonucleotide.
 12. The oligonucleotide of claim 1 wherein eachnucleotide of said first portion, independently, is a 2′-SCH₃ribonucleotide, a 2′-NH₂ ribonucleotide or a 2′-CH₃ ribonucleotide. 13.The oligonucleotide of claim 1 wherein each nucleotide of said firstportion is a 2′-SCH₃ ribonucleotide.
 14. The oligonucleotide of claim 1wherein each nucleotide of said first portion, independently, is a2′-SCH₃ ribonucleotide, a 2′-NH₂ ribonucleotide a 2′-NH(C₁-C₂alkyl)ribonucleotide, a 2′-N(C₁-C₂ alkyl)₂ ribonucleotide, a 2′-CH₃ribonucleotide, a 2′-CH═CH₂ ribonucleotide or a 2′-C≡CH ribonucleotide;and each nucleotide of said further portion, independently, is a 2′-Fribonucleotide, a 2′-O—(C₁-C₆ alkyl)ribonucleotide, or a 2′-O—(C₁-C₆substituted alkyl)ribonucleotide wherein the substitution is C₁-C₆ether, C₁-C₆ thioether, amino, amino(C₁-C₆ alkyl) or amino(C₁-C₆alkyl)₂.
 15. The oligonucleotide of claim 1 wherein said further portioncomprises at least two nucleotides joined together in a continuoussequence that is positioned at the 3′ terminus end of saidoligonucleotide.
 16. The oligonucleotide of claim 1 wherein said furtherportion comprises at least two nucleotides joined together in acontinuous sequence that is positioned at the 5′ terminus of saidoligonucleotide.
 17. The oligonucleotide of claim 1 wherein said furtherportion comprises at least two nucleotides joined together in acontinuous sequence that is positions at the 3′ terminus of saidoligonucleotide; and at least two nucleotides joined together in acontinuous sequence that is positions at the 5′ terminus of saidoligonucleotide.
 18. The oligonucleotide of claim 15 wherein said atleast two nucleotides joined together comprise nucleotides joinedtogether by a 2′-5′ phosphodiester linkage, a 3′-methylenephosphonatelinkage, a Sp phosphorothioate linkage, a methylene(methylimino)linkage, a dimethyhydrazino linkage, a 3′-deoxy-3′-aminophosphoroamidate linkage, an amide 3 linkage or an amide 4 linkage. 19.The oligonucleotide of claim 18 wherein said two nucleotides are joinedtogether by a 2′-5′ phosphodiester linkage, a 3′-methylenephosphonatelinkage, a Sp phosphorothioate linkage or a methylene(methylimino)linkage.
 20. The oligonucleotide of claim 16 wherein said at least twonucleotides joined together comprise nucleotides joined together by a2′-5′ phosphodiester linkage, a 3′-methylenephosphonate linkage, a Spphosphorothioate linkage, a methylene(methylimino) linkage, adimethyhydrazino linkage, a 3′-deoxy-3′-amino phosphoroamidate linkage,an amide 3 linkage or an amide 4 linkage.
 21. The oligonucleotide ofclaim 20 wherein said two nucleotides are joined together by a 2′-5′phosphodiester linkage, a 3′-methylenephosphonate linkage, a Spphosphorothioate linkage or a methylene(methylimino) linkage.
 22. Theoligonucleotide of claim 17 wherein said at least two nucleotides joinedtogether and positioned at said 3′ terminus comprise nucleotides joinedtogether by a 2′-5′ phosphodiester linkage, a 3′-methylenephosphonatelinkage, a Sp phosphorothioate linkage, a methylene(methylimino)linkage, a dimethyhydrazino linkage, a 3′-deoxy-3′-aminophosphoroamidate linkage, an amide 3 linkage or an amide 4 linkage; andwherein said at least two nucleotides joined together and positioned atsaid 5′ terminus comprise nucleotides joined together by a 2′-5′phosphodiester linkage, a 3′-methylenephosphonate linkage, a Spphosphorothioate linkage, a methylene(methylimino) linkage, adimethyhydrazino linkage, a 3′-deoxy-3′-amino phosphoroamidate linkage,an amide 3 linkage or an amide 4 linkage.
 23. The oligonucleotide ofclaim 22 wherein said two nucleotides joined together at said 3′terminus and said two nucleotides joined together at said 5′ terminusare, independently, joined together by 2′-5′ phosphodiester linkages,3′-methylenephosphonate linkages, Sp phosphorothioate linkages ormethylene(methylimino) linkages.
 24. The oligonucleotide of claim 15wherein at least one of said two nucleotides joined together is a2′-alkylamino substituted nucleotide.
 25. The oligonucleotide of claim16 wherein at least one of said two nucleotides joined together is a2′-alkylamino substituted nucleotide.
 26. The oligonucleotide of claim17 wherein at least one of said two nucleotides joined together at said3′ terminus is a 2′-alkylamino substituted nucleotide, and wherein atleast one of said two nucleotides joined together at said 5′ terminus isa 2′-alkylamino substituted nucleotide.